Compositions and methods for modulating il-10 immunostimulatory and anti-inflammatory properties

ABSTRACT

The invention provides compositions and methods for modulating the immunostimulatory properties and/or anti-inflammatory properties of IL-10. The present invention provides scIL-10 polypeptides of Formula 1. The polypeptides of the invention are optionally linked to a fusion partner. The polypeptides of Formula 1 are referred to herein as “scIL-10” polypeptides and comprise an amino acid sequence arrangement from N-terminus to C-terminus in accordance with Formula 1: 
       (first monomer subunit)-LINKER-(second monomer subunit)   Formula 1
         wherein the first monomer subunit, the second monomer subunit or both the first and second monomer subunits may be independently selected from: an unsubstituted IL-10 monomer subunit; or a substituted IL-10 monomer subunit comprising at least one amino acid substitution; and wherein LINKER is any amino acid linker of at least 1-100 amino acids in length.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/353,478, filed Jun. 22, 2016. The entire teachings of the aboveapplication(s) are incorporated herein by reference.

BACKGROUND OF THE INVENTION

IL-10 is considered a potent anti-inflammatory cytokine that stronglyinhibits the production of inflammatory mediators. However, recentstudies have suggested that IL-10 also has immunostimulatory propertieson CD4⁺, CD8⁺ T cells, and/or NK cells, resulting in increased IFN-γproduction which in turn may lead to related inflammatory responses inhumans.

Despite encouraging pre-clinical data suggesting this cytokine astherapeutically valuable biological, results of clinical trialsevaluating the merit of IL-10 administration in chronic inflammationhave been preponderantly disappointing. Bulk of pre-clinical data andanalysis of patients with IL-10 or IL-10 receptor defects clearly pointto endogenously produced IL-10 as potent and significantanti-inflammatory determinant. However, thorough analysis furthersuggests that IL-10 has the potential to acquire sharply contrastingproperties in an inflammatory environment in vivo. In recent yearsseveral studies have been performed in order to verify the humanresponse upon IL-10 administration, particularly in view of itsanti-inflammatory potential. Those clinically important studiesdisclosed perplexing pro-inflammatory functions of IL-10. However, thebasis of IL-10 immunostimulatory action remains unclear.

On the other hand, IL-10 has been explored for use in the treatment ofproliferative disorders, e.g., cancer, tumors, etc. IL-10 inducescytotoxic activity of CD8 T-cells, antibody production of B-cell andsuppresses macrophage activity and tumor promoting inflammation. IL-10appears to increase the infiltration of CD8+ T cells to a tumor, as wellas increasing the expression of inflammatory cytokines that play a rolein tumor immunity. Treatment with IL-10 may provide a significantimprovement for tumor treatment.

One drawback of using IL-10 and particularly any form of recombinantIL-10 in therapy is its short serum half-life. One strategy forincreasing serum half-life of a therapeutic protein such as IL-10 is toattach the protein to an Fc (fragment crystallizable) domain of anantibody. Many such fusion proteins are capable of forming homodimers orheterodimers thereby forming antibody-like fusion protein molecules.

Depending on the therapeutic application, the ability to selectivelyenhance either the anti-inflammatory activity or the immunostimulatoryactivity of IL-10 would be desired. It would also be desirable toincrease the half-life of recombinant IL-10.

SUMMARY OF THE INVENTION

The invention provides compositions and methods for modulating theimmunostimulatory properties and/or anti-inflammatory properties ofIL-10. The present invention provides scIL-10 polypeptides of Formula 1.The polypeptides of the invention are optionally linked to a fusionpartner. The polypeptides of Formula 1 are referred to herein as“scIL-10” polypeptides and comprise an amino acid sequence arrangementfrom N-terminus to C-terminus in accordance with Formula 1:

(first monomer subunit)-LINKER-(second monomer subunit)   Formula 1

wherein the first monomer subunit, the second monomer subunit or boththe first and second monomer subunits may be independently selectedfrom: an unsubstituted IL-10 monomer subunit; or a substituted IL-10monomer subunit comprising at least one amino acid substitution; andwherein LINKER is any amino acid linker of at least 1-100 amino acids inlength.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale emphasis instead being placed upon illustrating theprinciples of the invention.

FIG. 1 is a diagram of an Fc fusion protein homodimer of two polypeptidechains, wherein in each polypeptide chain comprises as X, scIL-10 whichis then fused to the Fc region of an IgG1 antibody via an scCLCH1linker.

FIG. 2 is a diagram of an Fc fusion protein homodimer of two polypeptidechains, wherein in each polypeptide chain comprises scIL-10 which isthen fused to the Fc region of an IgG1 antibody via the novel scCH1CLlinker.

FIG. 3 is an SDS-PAGE showing expression of an Fc fusion proteincomprising scIL-10 fused to the Fc region of an IgG1 antibody via thenovel scCLCH1 linker (left) or via the novel scCH1CL linker (right)under reducing and non-reducing conditions.

FIG. 4 is a chromatogram showing the characterization of the IL-10 fusedto the Fc region of an IgG1 antibody via the novel scCLCH1 linker byanalytical gel filtration.

FIG. 5 is a chromatogram showing the characterization of the IL-10 fusedto the Fc region of an IgG1 antibody via the novel scCH1CL linker byanalytical gel filtration.

FIG. 6 is a graph showing stimulation of mouse mast cell line MC/9 bythe IL-10 single chain fusion proteins of the invention as compared tothe scIL-10 direct Fc fusion protein used as a control.

FIG. 7 is a schematic of the effects of amino acid substitutions thatdisrupt either one or both of the two IL-10R1 interfaces (SEQ ID NOS:20, 21 and 22).

FIG. 8 is a schematic of the effects of amino acid substitutions thatdisrupt either one or both of the two IL-10R2 interfaces (SEQ ID NOS:23, 24 and 25).

FIG. 9 is a schematic of the effects of amino acid substitutions thatsimultaneously disrupt one of the IL-10R1 and one of the IL-10R2interfaces. (SEQ ID NOS: 26-29).

DETAILED DESCRIPTION OF THE INVENTION Definitions

By “polypeptide” is meant any sequence of two or more amino acids,regardless of length, post-translation modification, or function.“Polypeptide,” “peptide,” and “protein” are used interchangeably herein.Polypeptides can include natural amino acids and non-natural aminoacids. Polypeptides can also be modified in any of a variety of standardchemical ways (e.g., an amino acid can be modified with a protectinggroup; the carboxy-terminal amino acid can be made into a terminal amidegroup; the amino-terminal residue can be modified with groups to, e.g.,enhance lipophilicity; or the polypeptide can be chemically glycosylatedor otherwise modified to increase stability or in vivo half-life).Polypeptide modifications can include the attachment of anotherstructure such as a cyclic compound or other molecule to the polypeptideand can also include polypeptides that contain one or more amino acidsin an altered configuration (i.e., R or S; or, L or D).

As used herein, “antibody” and “immunoglobulin” are used interchangeablyand refer to a polypeptide substantially encoded by an immunoglobulingene or immunoglobulin genes, or fragments thereof, which specificallybind and recognize an antigen. Identified immunoglobulin genes includethe kappa, lambda, alpha, gamma, delta, epsilon and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.Terms understood by those in the art of antibody technology are eachgiven the meaning acquired in the art, unless expressly defineddifferently herein. Antibodies are known to have variable regions, ahinge region, and constant domains. Immunoglobulin structure andfunction are reviewed, for example, in Harlow et al, Eds., Antibodies: ALaboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, ColdSpring Harbor, 1988).

The invention provides functional variants of the fusion proteins ofFormulas 1 and 2 and functional portions thereof. A “functional variant”of a fusion protein of Formulas 1 and 2 as used herein refers to apolypeptide or protein having substantial or significant sequenceidentity to a polypeptide or protein of Formula 1 or Formula 2, whichfunctional variant retains the biological activity of the polypeptide ofFormula 1 or Formula 2 of which it is a variant. Functional variantsencompass for example, those variants of Formulas 1 and 2 that retainthe ability to recognize target cells and target receptors to a similarextent or the same extent, or a higher extent as compared to the peptideof Formula 1 or 2. For example an amino acid sequence encoding afunctional variant of a peptide of Formula 1 or Formula 2 can be about50% identical, about 60% identical, about 70% identical, about 80%identical about 90% identical, about 95% identical, about 98% identical,about 99% identical to the amino acid sequence of a peptide of Formula 1or 2.

The term “functional portion” of the peptides of Formulas 1 and 2 refersto any part or fragment of the peptides of Formulas 1 and 2 whichretains the biological activity of a peptide of Formulas 1 or 2 fromwhich the functional portion is derived. For example, a functionalportion of a peptide of Formula 1 or Formula 2 may comprise an aminoacid sequence comprising about 10%, 25%, 30%, 50%, 60%, 70%, 80%, 90% ormore of the parent peptide of Formula 1 or Formula 2. A functionalportion of a peptide of Formula 1 or Formula 1 may comprise additionalamino acids at the amino or carboxy terminus portion, or at bothtermini, which additional amino acids are not found in the amino acidsequence of the parent protein of Formula 1 or Formula 2. Preferably,the additional amino acids do not interfere with the biological functionof the functional portion.

Sequences similar or homologous (e.g., at least about 70% sequenceidentity) to the sequences disclosed herein are also part of theinvention. In some embodiments, the sequence identity at the amino acidlevel can be about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or higher. At the nucleic acid level, the sequenceidentity can be about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or higher. Alternatively, substantial identity existswhen the nucleic acid segments will hybridize under selectivehybridization conditions (e.g., very high stringency hybridizationconditions), to the complement of the strand. The nucleic acids may bepresent in whole cells, in a cell lysate, or in a partially purified orsubstantially pure form.

Calculations of “homology” or “sequence identity” or “similarity”between two sequences (the terms are used interchangeably herein) areperformed as follows. The sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, 90%, 100% of the length of thereference sequence. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “homology” is equivalent to aminoacid or nucleic acid “identity”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences. In the case of circularly related proteins, the sequence ofone of the partners needs to be appropriately split and aligned in twosections to achieve optimal alignment of the functionally equivalentresidues necessary to calculate the percent identity.

Amino acid and nucleotide sequence alignments and homology, similarityor identity, as defined herein are preferably prepared and determinedusing the algorithm BLAST 2 Sequences, using default parameters(Tatusova, T. A. et al., FEMS Microbiol Lett, 174:187-188 (1999)).Alternatively, the BLAST algorithm (version 2.0) is employed forsequence alignment, with parameters set to default values. BLAST (BasicLocal Alignment Search Tool) is the heuristic search algorithm employedby the programs blastp, blastn, blastx, tblastn, and tblastx; theseprograms ascribe significance to their findings using the statisticalmethods of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA87(6):2264-8.

The notations “mg/kg”, or “mg per kg” refer to milligrams per kilogram.All notations are used interchangeably throughout the presentdisclosure.

The “half-life” of a polypeptide can generally be defined as the timetaken for the serum concentration of the polypeptide to be reduced by50%, in vivo, for example due to degradation of the polypeptide and/orclearance or sequestration of the polypeptide by natural mechanisms. Thehalf-life can be determined in any manner known per se, such as bypharmacokinetic analysis. Suitable techniques will be clear to theperson skilled in the art, and may, for example, generally involve thesteps of administering a suitable dose of a polypeptide to a rodent orprimate; collecting blood samples or other samples from a rodent orprimate at regular intervals; determining the level or concentration ofthe polypeptide in said blood sample; and calculating, from (a plot of)the data thus obtained, the time until the level or concentration of thepolypeptide has been reduced by 50% compared to the initial level upondosing. Methods for determining half-life may be found, for example, inKenneth et al., Chemical Stability of Pharmaceuticals: A Handbook forPharmacists (1986); Peters et al, Pharmacokinete analysis: A PracticalApproach (1996); and “Pharmacokinetics”, M Gibaldi & D Perron, publishedby Marcel Dekker, 2nd Rev. edition (1982).

The half-life of a fusion polypeptide is increased if presence in abiological matrix (blood, serum, plasma, tissue) persists, in vivo, fora longer period as compared to an appropriate control. Half-life may beincreased by 10%, 20%, 30%, 40%, 50% or more as compared to anappropriate control.

Half-life can be expressed using parameters such as the t_(1/2-alpha),t_(1/2-beta), and HL_Lambda_z. In the present specification, an“increase in half-life” refers to an increase in any one of theseparameters, any two of these parameters, or all three of theseparameters. An “increase in half-life” in particular refers to anincrease in the t_(1/2)-beta and/or HL_Lambda_z, either with or withoutan increase in the t_(1/2)-alpha. Other PK parameters that can beassessed include volume of distribution (VD), clearance (CL), and meanresidence time (MRT), and the area under the curve (AUC). In the presentspecification, a “change in pharmacokinetics” refers to changes in anyone of these parameters, any two of these parameters, any three of theseparameters, or all four of these parameters, in the presence or absenceof changes in the half-life parameters listed above.

“Activity” for the purposes herein refers to an action or effect of acomponent of a fusion protein consistent with, but not necessarilyidentical to, that of the corresponding native active protein, wherein“biological activity” or “bioactivity” refers to an in vitro or in vivobiological function or effect, including but not limited to receptorbinding, antagonist activity, agonist activity, or a cellular orphysiologic response.

As used herein, a “dimer complex” comprises two single chains ofsc-IL-10, or sc-IL-10 fused to an appropriate fusion partner such as,for example, the scIL-10-L1-HINGE-Fc fusion proteins of the invention,wherein the two single chain polypeptides are associated together underappropriate conditions via either non-covalent binding or covalentbinding, for example, by a disulfide bridge. A “heterodimeric protein”,“heterodimerized complex”, or “heterodimer” as used interchangeablyherein refers to a protein that is made of two single chainscIL-10-L1-HINGE-Fc polypeptides forming a dimer complex, wherein saidtwo single chain polypeptides have different amino acid sequences. Forexample, one single chain peptide of the heterodimer has an scIL-10based on Formula 1 with at least one amino acid substitution and theother single chain peptide of the heterodimer has an scIL-10 sequencebased on Formula 1 with no amino acid substitutions. A “homodimericprotein” “homodimerized complex” or “homodimer” as used interchangeablyherein, refers to a protein that is made of two identical orsubstantially identical polypeptides forming the dimer complex, whereinsaid two single chain polypeptides preferably share 100% identity. Thereare circumstances, especially with regard to larger polypeptides whereina homodimer comprises two substantially identical polypeptides having atleast about 95% or at least about 99% identity, wherein any amino aciddifferences between the two polypeptide chains comprise amino acidsubstitutions, additions or deletions which do not affect the functionaland physical properties of the polypeptide compared to its partnerpolypeptide of the homodimer such as, for example, conservative aminoacid substitutions.

As used herein, a protein is “soluble” when it lacks any transmembranedomain or protein domain that anchors or integrates the polypeptide intothe membrane of a cell expressing such polypeptide.

As used herein, “Fc domain”, “Fc region” or “Fc portion” as those termsmay be used interchangeably herein to describe an scIL-10-L1-HINGE-Fcfusion protein of the invention, encompasses domains derived from theconstant region of an immunoglobulin, preferably a human immunoglobulin,including a fragment, analog, variant, mutant or derivative of theconstant region. Suitable immunoglobulins include IgG1, IgG2, IgG3,IgG4, and other classes such as IgA, IgD, IgE and IgM. The constantregion of an immunoglobulin is defined as a naturally-occurring orsynthetically-produced polypeptide homologous to the immunoglobulinC-terminal region, and can include a CH1 domain, a hinge, a CH2 domain,a CH3 domain, or a CH4 domain, separately or in combination.

As used herein, “treatment” or “treating,” or “palliating” or“ameliorating” is used interchangeably herein. These terms refer to anapproach for obtaining beneficial or desired results including but notlimited to a therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant eradication or amelioration of theunderlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the subject, notwithstanding that thesubject may still be afflicted with the underlying disorder.

For prophylactic benefit, the compositions may be administered to asubject at risk of developing a particular disease, or to a subjectreporting one or more of the physiological symptoms of a disease, eventhough a diagnosis of this disease may not have been made.

A “therapeutic effect”, as used herein, refers to a physiologic effect,including but not limited to the cure, mitigation, amelioration, orprevention of disease in humans or other animals, or to otherwiseenhance physical or mental well-being of humans or animals, caused by afusion protein of the invention.

The terms “therapeutically effective amount” and “therapeuticallyeffective dose”, as used herein, refers to an amount of an activeprotein, either alone or as a part of a fusion protein composition, thatis capable of having any detectable, beneficial effect on any symptom,aspect, measured parameter or characteristics of a disease state orcondition when administered in one or repeated doses to a subject. Sucheffect need not be absolute to be beneficial.

The term “therapeutically effective dose regimen”, as used herein,refers to a schedule for consecutively administered doses of an activeprotein, either alone or as a part of a fusion protein composition,wherein the doses are given in therapeutically effective amounts toresult in sustained beneficial effect on any symptom, aspect, measuredparameter or characteristics of a disease state or condition.

As used herein the “anti-inflammatory window” is defined as the range ofscIL-10 concentrations that produce anti-inflammatory effects onPBMCs/macrophages, while not inducing immunostimulatory effects (on CD8T cells, NK cells, etc. . . . ). For example, two assays are used in theExamples to define the potencies of those two bioactivities:

-   -   1) PBMC cytokine release assay: yields an IC50 value (usually in        the low picomolar range) for the concentration at which        anti-inflammatory effects occur as measured by inhibition of        release of TNF-alpha (TNFα); and    -   2) MC/9 proliferation assay: yields an EC50 value (usually in        the high picomolar to nanomolar range) for the concentration at        which immunostimulation effects occur.        The ratio in Tables 11 and 12 is the ratio of (MC/9 EC50)/(PBMC        IC50) values. These two assays represent an approximation of the        two types of activities. IL-10 targets cell populations within        PBMCs to suppress their release of pro-inflammatory cytokines        upon LPS stimulation, and IL-10 drives the proliferation of MC/9        cells at concentrations relevant to its immunostimulatory        effects. There are many other potential assays that may be used        to address the anti-inflammatory window size of the molecules of        the invention. However, it is understood that both the        immunostimulatory and anti-inflammatory effects of scIL-10 occur        in a wider number of cell types.        scIL-10

Human wild-type IL-10 (wtIL-10) is a non-covalently linked dimer proteincomprising two identical monomer subunits. Each identical monomersubunit of human wild type IL-10 (wtIL-10) has the following amino acidsequence (absent the leader sequence):

(SEQ ID NO: 1) SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMS EFDIFINYIEAYMTMKIRN(UniProtKB-P22301[chain 19-178] of IL 10, Interleukin-10, Homosapiens).SEQ ID NO: 1 is also referred to herein as an “unsubstituted IL-10monomer subunit”. Amino Acid sequences based on SEQ ID NO: 1 thatcomprise at least one amino acid substitution are referred to herein as“substituted IL-10 monomer subunits”. The invention also providessequences that are functional variants of portions of SEQ ID NO: 1 andsequences that are preferably at least 70% or more identical to SEQ IDNO: 1.

The polypeptides of Formula 1 are referred to herein as “scIL-10”polypeptides and comprise an amino acid sequence arrangement fromN-terminus to C-terminus in accordance with Formula 1:

(first monomer subunit)-LINKER-(second monomer subunit)   Formula 1

wherein the first monomer subunit, the second monomer subunit or boththe first and second monomer subunits may be independently selectedfrom: an unsubstituted IL-10 monomer subunit; or a substituted IL-10monomer subunit comprising at least one amino acid substitution; andwherein LINKER is any amino acid linker of at least 1-100 amino acids inlength.

Preferably, LINKER has a length of between at least 2 amino acid andless than 100 amino acids, such as for example between at least 2 aminoacids and less than 75 amino acids, more preferably between at least 3amino acids and less than 50 amino acids, such as for example between atleast 4 amino acids and less than 25 amino acids, such as for examplebetween at least 5 amino acids and less than 20 amino acids and evenmore preferably between at least 6 amino acids and less than 15 aminoacids. More preferably, the linker has a length of between at least 3amino acids and less than 10 amino acids. Most preferably, the linkerhas a length of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 aminoacids. Preferably, the linker is a flexible linker. Preferably, theflexible linker comprises or consists of the amino acids glycine,asparagine and/or serine. More preferably, the flexible linker comprisesor consists of the amino acids glycine and serine.

Preferably the first monomer subunit and the second monomer subunit ofFormula 1 are both unsubstituted IL-10 monomer subunits and each havethe amino acid sequence of SEQ ID NO: 1. These peptides are alsoreferred to herein as “unsubstituted sell-10”.

Preferably, scIL-10 peptides of Formula 1 comprise at least one aminoacid substitution in either the first monomer subunit of Formula 1, thesecond monomer subunit of Formula 1, or in both the first and secondmonomer subunits of Formula 1. These scIl-10 proteins comprisingsubstituted monomer subunits as compared to human wtscIL-10 of SEQ IDNO: 1 are also referred to herein as “scIL-10 variants”.

A preferred scIL-10 peptide of the invention is referred to herein as“unsubstituted scIL-10 (10aa linker)” and has the following amino acidsequence: SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGSGGGGSGGSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO:2) or a sequence thatis 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to(SEQ ID NO: 2). The ten amino acid linker between the two IL-10 subunitsat amino acids 179-188 is indicated by underlining. It is understoodthat other covalently linked IL-10 dimer proteins may include anysuitable flexible peptide linker and may also be longer or shorter thanthe underlined sequence of SEQ ID NO: 2.

scIL-10 (10aa linker) as represented by SEQ ID NO: 2 comprises twounsubstituted scIL-10 monomer subunits each comprising the amino acidsequence of SEQ ID NO: 1 and as per Formula 1, a LINKER, wherein LINKERis 10 amino acids in length having the sequence: GGSGGGGSGG (SEQ ID NO:3). Preferably LINKER of scIL-10 is not SEQ ID NO: 3 when the scIL-10peptide of the invention comprises two unsubstituted scIL-10 monomersubunits of SEQ ID NO: 1.

Other preferred unsubstituted scIL-10 peptides of Formula 1 includepeptides wherein LINKER is a 5 amino acid linker also referred to hereinas “unsubstituted sc-IL10 (5aa linker)”. One preferred five, amino acidlinker is the sequence: GGSGG (SEQ ID NO: 4).

Other preferred unsubstituted scIL-10 peptides of Formula 1 includepeptides wherein LINKER is a three amino acid linker also referred toherein as “unsubstituted sc-IL10 (3aa linker)”. One preferred three,amino acid linker is the sequence is the sequence GGG.

The present invention is based in part on the discovery that fusionproteins comprising unsubstituted scIL-10 and scIL-10 comprising atleast one amino acid substitution (“scIL-10 variants, possess a broadanti-inflammatory window. The present invention is also based in part onthe discovery that certain amino acid substitutions of unsubstitutedscIL-10 further increase the immunostimulatory EC₅₀. The ability toincrease the immunostimulatory EC₅₀ while maintaining a lowanti-inflammatory IC₅₀ provides several orders of magnitude increase inthe anti-inflammatory window size as compared to, for example, wild-typeIL-10 or other fusion proteins comprising IL-10 that are not modified inaccordance with the invention.

Without being limited to any theory, it is believed that amino acidsubstitutions at the interface of scIL-10 with the IL-10R1 and/orIL-10R2 receptor resulted in modulation of IL-10's immunostimulatoryproperties, anti-inflammatory properties or both.

It was found that an amino acid substitution at aspartic acid atposition 41 (based on SEQ ID NO: 1) in the first monomer subunit or ataspartic acid at position 41 (based on SEQ ID NO: 1) of the secondmonomer subunit of scIL-10 of Formula 1 disrupts at least one of thescIL-10 interfaces with its IL-10R1 receptor thereby slightly weakeningthe anti-inflammatory potency while significantly weakening theimmunostimulatory potency of scIL-10 resulting in an increase in theanti-inflammatory window. It was also found that mutations that disruptscIL-10 at one interface with IL-10R1 on one of either the first orsecond monomer subunit and also disrupts scIL-10 at one interface withIL-10R2, (for example at the methionine at position 22 of SEQ ID NO: 1)on either the first or second monomer subunit that is not the same asthe mutation that disrupts the IL-10R1 interface provides an extremelylarge anti-inflammatory window.

It was also discovered that an amino acid substitution of isoleucine atposition 87 (based on SEQ ID NO: 1) and which is believed to affect thebinding to both IL-10R1 and IL-10R2 appears to have a similar effect aswhen scIL-10 is designed to disrupt IL-10R1 in one subunit and disruptIL-10R2 in the other subunit. Without being limited to any theory, it isbelieved that the isoleucine at position 87 in human wtIL-10 modulatesthe interaction with both IL-10 receptors. See also, Ding et al, (2000)J. Exp. Med. 191(2):213.

Preferably, the invention provides scIL-10 variants wherein at least oneamino acid substitution (as compared to human wild type IL-10 of SEQ IDNO: 1) is introduced in the first and/or second monomer subunit ofFormula 1. Preferably scIL-10 comprises at least one amino acidsubstitution at the interface of the IL-10R1 interface on only one ofthe first or second monomer subunits of Formula 1 but not both of thefirst or second monomer subunits of Formula 1. Even more preferablyscIL-10 comprises at least one amino acid substitution at the interfaceof the IL-10R1 interface of only one of the first or second monomersubunits of Formula 1 and also comprises at least one amino acidsubstitution at an IL-10R2 interface on only one of the first or secondmonomer subunits of Formula that is not the same monomer subunit as theamino acid substitution at the IL-1-R1 interface.

Preferred amino acid substitutions for scIL-10 variants are based on thenumbering of amino acids of SEQ ID NO: 1 and include the followingmutations: methionine at position 22 and aspartic acid at position 41.

Preferably the invention provides scIL-10 variants wherein at least oneamino acid is substituted at position 41 in the first or second monomersubunit of Formula 1 and at least one amino acid is substituted atposition 22 in the first or second monomer subunit that is not the samesubunit that comprises the amino acid substitution at position 41.

Preferably the invention provides scIL-10 variants wherein at least oneamino acid is substituted at the isoleucine at position 87 of only thefirst monomer subunit or the second monomer subunit of Formula 1 but notat both monomer subunits.

Amino acid substitutions of methionine at position 22, aspartic acid atposition 41 and isoleucine at position 87 may include substitution withany other amino acid. Either conservative or non-conservative amino acidsubstitutions can be made at one or more amino acid residues. Bothconservative and non-conservative substitutions can be made.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains. Genetically encodedamino acids can be divided into four families: (1) acidic=aspartate,glutamate; (2) basic=lysine, arginine, histidine; (3) nonpolar(hydrophobic)=cysteine, alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan, glycine, tyrosine; and (4)uncharged polar=asparagine, glutamine, serine, threonine. Non-polar maybe subdivided into: strongly hydrophobic=alanine, valine, leucine,isoleucine, methionine, phenylalanine and moderatelyhydrophobic=glycine, proline, cysteine, tyrosine, tryptophan. Inalternative fashion, the amino acid repertoire can be grouped as (1)acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine, (3)aliphatic=glycine, alanine, valine, leucine, isoleucine, serine,threonine, with serine and threonine optionally be grouped separately asaliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine, tryptophan;(5) amide=asparagine, glutamine; and (6) sulfur-containing=cysteine andmethionine.

Preferred amino acid substitutions for the first monomer subunit and/orthe second monomer subunit in accordance with Formula 1 include thefollowing substitutions: the methionine at position 22 to alanine(M22A); aspartic acid at position 41 to asparagine (D41N); aspartic acidat position 41 to alanine (D41A); aspartic acid at position 41 tophenylalanine (D41F); isoleucine at position 87 to alanine (I87A).

The invention is also based in part on the discovery that theimmunostimulatory or anti-inflammatory activities of scIL-10 and scIL-10variants can be further modulated by fusing scIL-10 or scIL-10 variantsto fusion partners including, but not limited to, Fc polypeptides andmodified Fc polypeptides such as single chain Fc fusion proteins, mucinlinker Fc fusions, Fc polypeptides with truncated hinge regions. Otherfusion partners include, but are not limited to: mucin domainpolypeptides, albumin fusion proteins, transferrin proteins and otherfusion partners not comprising an Fc domain.

Single Chain Fc Fusion Proteins of sc-IL10

Single chain Fc fusion proteins of the invention have the followingarrangement from amino-terminus (N-terminus) to carboxy-terminus(C-terminus) as shown in Formula 2:

(scIL-10)-L1-HINGE:Fc   (Formula 2)

wherein, scIL-10 has the amino acid sequence of Formula 1;L1 is a linker having the following arrangement from amino-terminus tocarboxy-terminus:

L2-CL-L3-CH1-L4  (Formula 3) or

L2-CH1-L3-CL-L4  (Formula 4)

-   -   wherein,    -   L2 and L4 are independently polypeptide linkers or are        independently absent,    -   L3 is a polypeptide linker;    -   CL is a constant region polypeptide from an immunoglobulin light        chain; and    -   CH1 a constant region polypeptide from a CH1 domain of an        immunoglobulin heavy chain;        HINGE is a hinge sequence of an immunoglobulin or is absent with        the proviso that if HINGE is absent, L4 is present; and Fc is        the carboxy-terminus of an immunoglobulin or any active fragment        or derivative thereof.

In accordance with the invention, an scIL-10 of Formula 1 is fused tothe N-terminal region of an immunoglobulin Fc region via a novel linker(L1) that is derived from the CL and CH1 domains of an immunoglobulinarranged as a single chain (sc) also referred to herein as “scCLCH1linkers” (Formula 3).

The C-terminus of the CL region may be linked to the N-terminal regionof a CH1 region via polypeptide linker L3. The N-terminus of the CLregion may be fused to the C-terminus of scIL-10 of Formula 1 via anoptional polypeptide linker L2. The C-terminus of the CH1 domain islinked to the Fc domain via an immunoglobulin hinge region (HINGE) or apolypeptide linker (L4) or both a hinge (HINGE) and a polypeptide linker(L4).

The C-terminus of the CH1 domain may also be linked to the N-terminus ofa CL region via polypeptide linker L3. The N-terminus of the CH1 regionmay be fused to the C-terminus of scIL-10 of Formula 1 via an optionalpolypeptide linker L2. The C-terminus of the CL region is linked to theFc region via an immunoglobulin hinge region (HINGE) or a polypeptidelinker (L4) or both a hinge (HINGE) and a polypeptide linker (L4).

Preferably, L3 is selected from artificial flexible domains comprisingamino acids selected from Gly (G), and/or Ser (S). Preferably, thelinker is comprised of polypeptide of the general formula(Gly-Gly-Gly-Ser (SEQ ID NO: 5))n or (Gly-Gly-Gly-Gly-Ser (SEQ ID NO:6))n wherein n is an integer from 1 to 10. Preferably, each linker is apolypeptide comprising from about 1 to about 100 amino acids, preferablyabout 1-50 amino acids, preferably about 1-25 amino acids, preferablyabout 1-15 amino acids preferably about 1-10 amino acids, preferablyabout 4-24 amino acids, preferably about 5-20 amino acids preferablyabout 5-15 amino acids and preferably about 5-10 amino acids.Preferably, the linker is (Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 6)) n whereinn is 2 or 4.

L2 and L4 are independently selected from artificial flexible domainscomprising amino acids selected from, for example, Gly (G), and Ser (S).Preferably, the linker is comprised of polypeptide of the generalformula (Gly-Gly-Gly-Ser (SEQ ID NO: 5))n or (Gly-Gly-Gly-Gly-Ser (SEQID NO: 6))n wherein n is an integer from 1 to 10. Preferably, eachlinker is a polypeptide comprising from about 1 to about 100 aminoacids, preferably about 1-50 amino acids, preferably about 1-25 aminoacids, preferably about 1-15 amino acids preferably about 1-10 aminoacids, preferably about 4-24 amino acids, preferably about 5-20 aminoacids preferably about 5-15 amino acids and preferably about 5-10 aminoacids. Preferably, the linker is (Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 6))nwherein n is 2 or 4.

L2, L3 and L4, may further comprise amino acids such as, for example,Lys (K), Thr (T), Glu (E), and Asp (D).

The CL region of the novel scCLCH1 linker (L1) may be substantiallyidentical to the corresponding CL region of native immunoglobulinsbelonging to any of the immunoglobulin classes, i.e., IgA, IgD, IgE,IgG, or IgM or any of the IgG antibody subclasses, i.e., IgG1, IgG2,IgG3, and IgG4. The CL region (L1) may have amino acid sequence that isat least 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the corresponding CL region of native immunoglobulinsbelonging to any of the immunoglobulin classes, i.e., IgA, IgD, IgE,IgG, or IgM or any of the IgG antibody subclasses, i.e., IgG1, IgG2,IgG3, and IgG4. If the CL region of L1 is a modified derivative orvariant of a native CL region such modifications include, but are notlimited to, amino acid insertions, deletions, substitutions andrearrangements. Preferably, the amino acid sequence of the CL region inaccordance with the invention, is at least 80%, more preferably at least85%, more preferably at least 90%, and more preferably at least 95%identical to the corresponding CL region of native immunoglobulinsbelonging to any of the immunoglobulin classes, i.e., IgA, IgD, IgE,IgG, or IgM or any of the IgG antibody subclasses, i.e., IgG1, IgG2,IgG3, and IgG4.

The CH1 region of the novel scCLCH1 linker (L1) may be substantiallyidentical to the corresponding CH1 region of native immunoglobulinsbelonging to any of the immunoglobulin classes, i.e., IgA, IgD, IgE,IgG, or IgM or any of the IgG antibody subclasses, i.e., IgG1, IgG2,IgG3, and IgG4. The CH1 region of L1 may have amino acid sequence thatis at least 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the corresponding CH1 region of native immunoglobulinsbelonging to any of the immunoglobulin classes, i.e., IgA, IgD, IgE,IgG, or IgM or any of the IgG antibody subclasses, i.e., IgG1, IgG2,IgG3, and IgG4. If the CH1 region of the L1 linker is a modifiedderivative or variant of a native CH1 immunoglobulin region suchmodifications include, but are not limited to, amino acid insertions,deletions, substitutions and rearrangements. Preferably, the amino acidsequence of the CH1 region is at least 80%, more preferably at least85%, more preferably at least 90%, and more preferably at least 95%identical to the corresponding CH1 region of native immunoglobulinsbelonging to any of the immunoglobulin classes, i.e., IgA, IgD, IgE,IgG, or IgM or any of the IgG antibody subclasses, i.e., IgG1, IgG2,IgG3, and IgG4.

The CH1 region and CL regions of L1 do not need to be identical to or avariant of, the corresponding regions of the same immunoglobulin class.For example, the CL region may be derived from the corresponding regionof IgE and the CH1 region may be derived from the corresponding regionof IgG.

Preferably, CL and CH1 of the scCLCH1 linker are derived from thecorresponding CL and CH1 regions of IgG1, preferably human IgG1.

An exemplary CL region corresponding to the CL region of a human IgG1(hIgG1) includes:

(SEQ ID NO: 7) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGES.

An exemplary CH1 region corresponding to the CH1 region of hIgG1includes:

(SEQ ID NO: 8) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKRV.

The single chain Fc fusion proteins disclosed herein comprise an Fcregion that includes at least a portion of the carboxy-terminus of animmunoglobulin heavy chain. For example, the Fc portion may comprise: aCH2 domain, a CH3 domain, a CH4 domain, a CH2-CH3 domain, a CH2-CH4domain, a CH2-CH3-CH4 domain, a hinge-CH2 domain, a hinge-CH2-CH3domain, a hinge-CH2-CH4 domain, or a hinge-CH2-CH3-CH4 domain. The Fcdomain may be derived from antibodies belonging any of theimmunoglobulin classes, i.e., IgA, IgD, IgE, IgG, or IgM or any of theIgG antibody subclasses, i.e., IgG1, IgG2, IgG3, and IgG4. Preferably,the Fc region is derived from IgG1 preferably human IgG1.

The Fc domain may be a naturally occurring Fc sequence belonging any ofthe immunoglobulin classes, i.e., IgA, IgD, IgE, IgG, or IgM or any ofthe IgG antibody subclasses, i.e., IgG1, IgG2, IgG3, and IgG4, includingnatural allelic or splice variants. Alternatively, the Fc domain may bea hybrid domain comprising a portion of an Fc domain from two or moredifferent Ig isotypes, for example, an IgG2/IgG4 hybrid Fc domain.Preferably, the Fc domain is derived from a human immunoglobulinmolecule. Alternatively, the Fc domain may be a humanized or deimmunized(removal of T cell epitopes which can activate helper T cells) versionof an Fc domain from a non-human animal, including but not limited tomouse, rat, rabbit, and monkey.

The Fc domain may be a variant Fc sequence, e.g., an Fc sequence thathas been modified (e.g., by amino acid substitution, deletion and/orinsertion) relative to a parent Fc sequence (e.g., an unmodified Fcpolypeptide that is subsequently modified to generate a variant), toprovide desirable structural features and/or biological activity. Forexample, one may make modifications in the Fc region in order togenerate an Fc variant that (a) has increased or decreasedantibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased ordecreased complement mediated cytotoxicity (CDC), (c) has increased ordecreased affinity for C1q and/or (d) has increased or decreasedaffinity for a Fc receptor relative to the parent Fc. Such Fc regionvariants will generally comprise at least one amino acid modification inthe Fc region. Combining amino acid modifications is thought to beparticularly desirable. For example, the variant Fc region may includetwo, three, four, five, etc. substitutions therein, e.g. of the specificFc region positions identified herein.

The hinge region of the Fc fusion proteins of the invention may bederived from antibodies belonging to any of the immunoglobulin classes,i.e., IgA, IgD, IgE, IgG, or IgM. The hinge region may be derived fromany of the IgG antibody subclasses, i.e., IgG1, IgG2, IgG3, and IgG4.The hinge region may naturally contain a cysteine residue or may beengineered to contain one or more cysteine residues.

Preferably, the hinge region may have an amino acid sequence that is atleast 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identicalto the corresponding hinge region of native immunoglobulins belonging toany of the immunoglobulin classes, i.e., IgA, IgD, IgE, IgG, or IgM orany of the IgG antibody subclasses, i.e., IgG1, IgG2, IgG3, and IgG4.Preferably, the amino acid sequence of the hinge region is at least 80%,more preferably at least 85%, more preferably at least 90%, and morepreferably at least 95% identical to the corresponding hinge region ofhuman IgG1.

Shown below is the sequence of a human IgG1 immunoglobulin constantregion, and the relative position of the hinge region is indicated bysolid underlining:

(SEQ ID NO: 9) 

VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.The CH1 region is indicated by underlining with a dotted line, and theCH2 and CH3 regions are indicated by bold lettering. The C-terminallysine of an IgG sequence may be removed or replaced with a non-lysineamino acid, such as alanine, to further increase the serum half-life ofthe Fc fusion protein.

The hinge sequence may include substitutions that confer desirablepharmacokinetic, biophysical, and/or biological properties. An exemplaryhinge region of the invention comprises an amino acid sequence that isat least 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the following: EPKSSDKTHTCPPCP (SEQ ID NO: 51).

The Fc domain and the hinge region may be derived from one antibodyclass or subclass. For example, the hinge region and the Fc domain maybe derived from IgG1. The Fc domain and hinge region may correspond todifferent antibody classes or subclasses. For example, the Fc domain maycorrespond to the Fc region of IgG2 or IgG4 and the hinge region maycorrespond to IgG1.

Preferably, all immunoglobulin domains of the Fc fusion proteins of theinvention are derived from IgG1, preferably human IgG1. Preferably, thecombined hinge region and Fc region of the fusion proteins of theinvention comprise an amino acid sequence that is at least 50%, 60%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to:EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10).Preferably, the combined hinge region and Fc region of the fusionproteins of the invention comprise an amino acid sequence that is atleast 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identicalto:

(SEQ ID NO: 11) EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPQVKFNWYVDGVQVHNAKTKPREQQYNSTYRVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

It may be desirable to have a hinge sequence and/or Fc region of thesingle chain fusion proteins of the invention comprising a free cysteineresidue in order to permit the formation of a disulfide bond between thehinge and or Fc regions thereby forming dimers of the Fc fusion proteinsof the invention. It may be desirable to alter the hinge and/or Fcregion sequences to remove free cysteine residues, e.g., by mutating oneor more cysteine residues in a linker to another residue, such as aserine, alanine or glycine. The hinge region of the single chain fusionproteins of the invention may comprise one or more free cysteineresidues capable of forming one or more disulfide bonds with a secondsingle chain fusion protein of the invention thereby forming a dimercomplex.

Preferably, the (scIL-10)-L1-HINGE-Fc fusion proteins of the inventionare dimer complexes comprising two monomeric single chain(scIL-10)-L1-HINGE-Fc fusion proteins of the invention linked via adisulfide bond to the hinge region or in the Fc region of the othermonomer. The dimer complexes may be homodimeric (e.g., both monomericfusion proteins are identical) or heterodimeric (e.g., scIL-10 may bedifferent for each monomeric fusion protein). Preferably, the dimercomplexes are homodimers thereby forming a homodimeric complex thatprovides an antibody configuration that resembles that of a nativeantibody.

Without being limited to any one theory, it is believed that thehomodimeric fusion proteins of the invention increase half-life due tothe presence of a dimerized Fc region which more closely resembles thenative antibody structure as compared to traditional Fc fusion proteins.This is particularly true when the fusion protein has the configurationof Formula 3. A more native Fc domain antibody configuration is believedto enable better binding to the FcRn receptor and therefore increase thecirculating half-life of the of the scIL-10-L1-HINGE-Fc dimer complex.

Another improved property associated with scIL-10-L1-HINGE-Fc dimercomplexes is that bioactivity is increased versus a traditional Fcfusion proteins based on the use of the scCLCH1 linker which impartsflexibility to relieve steric hindrance caused by the dimerizationthrough the Fc in the hinge region.

Preferably the invention provides (scIL-10)-L1-HINGE-Fc fusion whereinscIL-10 of Formula 1 is unsubstituted scIL-10 (10aa linker). Preferablythe invention provides (scIL-10)-L1-HINGE-Fc fusion wherein the scIL-10of Formula 1 is an sc-IL-10 variant comprising at least one amino acidsubstitution in the first monomer subunit or the second monomer subunitas per Formula 1 selected from the methionine at position 22, theaspartic acid at position 41, and the isoleucine at position 87 or anycombination thereof. Preferably there is at least one amino acidsubstitution at position 41 in the first or second monomer subunit ofFormula 1 and at least one amino acid is substituted at position 22 inthe first or second monomer subunit that is not the same subunit thatcomprises the amino acid substitution at position 41.

A preferred scIL10-L1-HINGE-Fc fusion protein of the invention comprisesan amino acid sequence that is 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to SEQ ID NO: 12 wherein scIL-10 isunsubstituted scIL-10 (10aa linker).

Preferred scIL-10-L1-HINGE-Fc fusion proteins of the invention comprisean amino acid sequence that is 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to SEQ ID NOs: 20-21 and 37-44 all as shownin Table 4.

Preferred scIL-10-L1-HINGE-Fc fusion proteins of the invention comprisean amino acid sequence that is 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to SEQ ID NOs: 17-19 as shown in Table 4wherein scIL-10 is an scIL-10 variant.

The invention also provides nucleic acids encoding any of the variousfusion proteins disclosed herein. Codon usage may be selected so as toimprove expression in a cell. Such codon usage will depend on the celltype selected. Specialized codon usage patterns have been developed forE. coli and other bacteria, as well as mammalian cells, plant cells,yeast cells and insect cells. See for example: Mayfield et al., Proc.Natl. Acad. Sci. USA, 100(2):438-442 (Jan. 21, 2003); Sinclair et al.,Protein Expr. Purif., 26(I):96-105 (October 2002); Connell, N. D., Curr.Opin. Biotechnol., 12(5):446-449 (October 2001); Makrides et al.,Microbiol Rev., 60(3):512-538 (September 1996); and Sharp et al., Yeast,7(7):657-678 (October 1991).

General techniques for nucleic acid manipulation are described forexample in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEdition, Vols. 1-3, Cold Spring Harbor Laboratory Press (1989), orAusubel, F. et al., Current Protocols in Molecular Biology, GreenPublishing and Wiley-Interscience, New York (1987) and periodic updates,herein incorporated by reference. Generally, the DNA encoding thepolypeptide is operably linked to suitable transcriptional ortranslational regulatory elements derived from mammalian, viral, orinsect genes. Such regulatory elements include a transcriptionalpromoter, an optional operator sequence to control transcription, asequence encoding suitable mRNA ribosomal binding sites, and sequencesthat control the termination of transcription and translation. Theability to replicate in a host, usually conferred by an origin ofreplication, and a selection gene to facilitate recognition oftransformants is additionally incorporated.

The fusion proteins described herein may be produced recombinantly notonly directly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected preferably isone that is recognized and processed (i.e., cleaved by a signalpeptidase) by the host cell. An exemplary N-terminal leader sequence forproduction of polypeptides in a mammalian system is MYRMQLLSCIALSLALVTNS(SEQ ID NO: 48), which is removed by the host cell following expression.

For prokaryotic host cells that do not recognize and process a nativesignal sequence, the signal sequence is substituted by a prokaryoticsignal sequence selected, for example, from the group of the alkalinephosphatase, penicillinase, or heat-stable enterotoxin II leaders.

For yeast secretion the native signal sequence may be substituted by,e.g., the yeast invertase leader, a factor leader (includingSaccharomyces and Kluyveromyces alpha-factor leaders), or acidphosphatase leader, the C. albicans glucoamylase leader, or the signaldescribed in U.S. Pat. No. 5,631,144. In mammalian cell expression,mammalian signal sequences as well as viral secretory leaders, forexample, the herpes simplex gD signal, are available. The DNA for suchprecursor regions may be ligated in reading frame to DNA encoding theprotein.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2 micron plasmid origin is suitablefor yeast, and various viral origins (SV40, polyoma, adenovirus, VSV orBPV) are useful for cloning vectors in mammalian cells. Generally, theorigin of replication component is not needed for mammalian expressionvectors (the SV40 origin may typically be used only because it containsthe early promoter).

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid encoding the protein disclosed herein, e.g., a fibronectin-basedscaffold protein. Promoters suitable for use with prokaryotic hostsinclude the phoA promoter, beta-lactamase and lactose promoter systems,alkaline phosphatase, a tryptophan (trp) promoter system, and hybridpromoters such as the tan promoter. However, other known bacterialpromoters are suitable. Promoters for use in bacterial systems also willcontain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding the protein disclosed herein. Promoter sequences are known foreukaryotes. Virtually all eukaryotic genes have an AT-rich regionlocated approximately 25 to 30 bases upstream from the site wheretranscription is initiated. Another sequence found 70 to 80 basesupstream from the start of transcription of many genes is a CNCAAT (SEQID NO: 49) region where N may be any nucleotide. At the 3′ end of mosteukaryotic genes is an AATAAA (SEQ ID NO: 50) sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Transcription from vectors in mammalian host cells can be controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovinepapilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus and most preferably Simian Virus 40 (SV40), fromheterologous mammalian promoters, e.g., the actin promoter or animmunoglobulin promoter, from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

Transcription of a DNA encoding proteins disclosed herein by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, α-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature, 297:17-18 (1982) on enhancingelements for activation of eukaryotic promoters. The enhancer may bespliced into the vector at a position 5′ or 3′ to the peptide-encodingsequence, but is preferably located at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (e.g., yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of mRNA encoding the protein disclosed herein.One useful transcription termination component is the bovine growthhormone polyadenylation region. See WO 94/11026 and the expressionvector disclosed therein.

The recombinant DNA can also include any type of protein tag sequencethat may be useful for purifying the protein. Examples of protein tagsinclude but are not limited to a histidine tag, a FLAG tag, a myc tag,an HA tag, or a GST tag. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts can befound in Cloning Vectors: A Laboratory Manual, (Elsevier, New York(1985)), the relevant disclosure of which is hereby incorporated byreference.

The expression construct is introduced into the host cell using a methodappropriate to the host cell, as will be apparent to one of skill in theart. A variety of methods for introducing nucleic acids into host cellsare known in the art, including, but not limited to, electroporation;transfection employing calcium chloride, rubidium chloride, calciumphosphate, DEAE-dextran, or other substances; microprojectilebombardment; lipofection; and infection (where the vector is aninfectious agent).

Suitable host cells include prokaryotes, yeast, mammalian cells, orbacterial cells. Suitable bacteria include gram negative or grampositive organisms, for example, E. coli or Bacillus spp. Yeast,preferably from the Saccharomyces species, such as S. cerevisiae, mayalso be used for production of polypeptides. Various mammalian or insectcell culture systems can also be employed to express recombinantproteins. Baculovirus systems for production of heterologous proteins ininsect cells are reviewed by Luckow et al. (Bio/Technology, 6:47(1988)). Examples of suitable mammalian host cell lines includeendothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3,Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293,293T, and BHK cell lines. Purified polypeptides are prepared byculturing suitable host/vector systems to express the recombinantproteins. For many applications, the small size of many of thepolypeptides disclosed herein would make expression in E. coli as thepreferred method for expression. The protein is then purified fromculture media or cell extracts.

In other aspects, the invention provides host cells containing vectorsencoding the fusion proteins described herein, as well as methods forproducing the fusion proteins described herein. Host cells may betransformed with the herein-described expression or cloning vectors forprotein production and cultured in conventional nutrient media modifiedas appropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences. Host cells usefulfor high-throughput protein production (HTPP) and mid-scale productioninclude the HMS 174-bacterial strain. The host cells used to produce theproteins disclosed herein may be cultured in a variety of media.Commercially available media such as Ham's F10 (Sigma), MinimalEssential Medium ((MEM), (Sigma)), RPMI-1640 (Sigma), and Dulbecco'sModified Eagle's Medium ((DMEM), Sigma)) are suitable for culturing thehost cells. In addition, many of the media described in variousscientific literature may be used as culture media for the host cells.Any of these media may be supplemented as necessary with hormones and/orother growth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics (such as Gentamycin drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

The fusion proteins provided herein can also be produced usingcell-translation systems. For such purposes the nucleic acids encodingthe fusion protein must be modified to allow in vitro transcription toproduce mRNA and to allow cell-free translation of the mRNA in theparticular cell-free system being utilized (eukaryotic such as amammalian or yeast cell-free translation system or prokaryotic such as abacterial cell-free translation system).

The fusion proteins disclosed herein can also be produced by chemicalsynthesis (e.g., by the methods described in Solid Phase PeptideSynthesis, 2nd Edition, The Pierce Chemical Co., Rockford, Ill. (1984)).Modifications to the fusion proteins can also be produced by chemicalsynthesis.

The fusion proteins disclosed herein can be purified byisolation/purification methods for proteins generally known in the fieldof protein chemistry. Non-limiting examples include extraction,recrystallization, salting out (e.g., with ammonium sulfate or sodiumsulfate), centrifugation, dialysis, ultrafiltration, adsorptionchromatography, ion exchange chromatography, hydrophobic chromatography,normal phase chromatography, reversed-phase chromatography, getfiltration, gel permeation chromatography, affinity chromatography,electrophoresis, countercurrent distribution or any combinations ofthese. After purification, polypeptides may be exchanged into differentbuffers and/or concentrated by any of a variety of methods known to theart, including, but not limited to, filtration and dialysis.

The purified fusion protein is preferably at least 85% pure, orpreferably at least 95% pure, and most preferably at least 98% pure.Regardless of the exact numerical value of the purity, the fusionprotein is sufficiently pure for use as a pharmaceutical product.

Other Fusion Partners

Other appropriate fusion partners for scIL-10 proteins of the inventioninclude but are not limited to proteins comprising an Fc region of allother types.

For example, scIL-10 proteins may be fused directly to the hinge regionof a native immunoglobulin containing an Fc region, for example IgG1.SEQ ID NO: 13 is an example of unsubstituted scIL-10 (5aa linker) fusedto the hinge region of an IgG1 molecule. The IgG1 molecule may bemodified, by, for example, by shortening the hinge region of IgG1. SEQID NO: 14 is an example of scIL-10 (5aa linker) fused to the hingeregion of IgG1 wherein in the hinge region of the native IgG1 has beenshortened by 4 amino acids. SEQ ID NO: 15 is an example of scIL-10 fusedto the hinge region of IgG1 wherein in the hinge region of the nativeIgG1 has been shortened by 7 amino acids. SEQ ID NO: 16 is an example ofscIL-10 fused to the hinge region of IgG1 wherein in the hinge region ofthe native IgG1 has been shortened by 10 amino acids.

A preferred fusion partner comprises an Fc region further comprising amucin-domain polypeptide linker as is described in WO 2013/184938incorporated herein by reference. A “mucin-domain polypeptide linker” isdefined herein as any protein comprising a “mucin domain” capable ofbeing linked to one or more fusion polypeptide partners. A mucin domainis rich in potential glycosylation sites, and has a high content ofserine and/or threonine and proline, which can represent greater than40% of the amino acids within the mucin domain. A mucin domain isheavily glycosylated with predominantly O-linked glycans. A mucin-domainpolypeptide has at least about 60%, at least 70%. at least 80%, or atleast 90% of its mass due to the glycans. Mucin domains may comprisetandem amino acid repeat units (also referred to herein as TR) that mayvary in length from about 8 amino acids to 150 amino acids per eachtandem repeat unit. The number of tandem repeat units may vary between 1and 25 in a mucin-domain polypeptide of the invention.

Mucin-domain polypeptide linkers of the invention include, but are notlimited to, all or a portion of a mucin protein. A “portion thereof” ismeant that the mucin polypeptide linker comprises at least one mucindomain of a mucin protein. Mucin proteins include any protein encodedfor by a MUC gene (e.g., MUC1, MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B,MUC6, MUC7, MUC8, MUC9, MUC11, MUC12, MUC13, MUC15, MUC16, MUC17, MUC19,MUC20, MUC21). The mucin domain of a mucin protein is typically flankedon either side by non-repeating amino acid regions. A mucin-domainpolypeptide may comprise all or a portion of a mucin protein (e.g.MUC20). A mucin-domain polypeptide may comprise all or a portion of amucin protein of a soluble mucin protein. Preferably the mucin-domainpolypeptide comprises the extracellular portion of a mucin protein.

Preferably, an scIL-10 protein of Formula 1 is covalently linked to amolecule comprising an Fc region via a mucin-domain polypeptide linker.SEQ ID NO: 52 is an example of unsubstituted scIL-10 fused to mucinlinker which is in turn fused to the hinge of a native IgG1 Fc region.

A preferred fusion partner is a mucin domain polypeptide (not includingan Fc region) as is described in WO 2013/184939.

A preferred fusion partner comprises serum albumin or a domain of serumalbumin. Human serum albumin is preferred when the fusion proteins ofthe invention are used for treating humans. In another embodiment,fusion partners comprise human transferrin.

Uses of scIL-10 Proteins

In one aspect, the invention provides scIL-10 (including fusions ofscIL-10 to an appropriate fusion partner and dimerized complexesthereof) that are useful as diagnostic or therapeutic agents. In oneaspect, the invention provides proteins useful in the treatment ofdisorders.

The invention also provides a method for achieving a beneficial effectin a subject comprising the step of administering to the subject atherapeutically or prophylactically-effective amount of scIL-10(including fusions of scIL-10 to an appropriate fusion partner anddimerized complexes thereof) of the invention. The effective amount canproduce a beneficial effect in helping to treat a disease or disorder.In some cases, the method for achieving a beneficial effect can includeadministering a therapeutically effective amount of a fusion proteincomposition to treat a subject for diseases and disease categorieswherein a therapeutic protein or peptide does not exist.

Preferably scIL-10 is not linked to any fusion partner.

Preferably, scIL-10 is covalently linked to an appropriate fusionpartner such as scIL-10-L1-HINGE-Fc. Preferably, the invention providesdimer complexes of scIL-10 fused to an appropriate fusion partner.

Preferably scIL-10 (including fusions of scIL-10 to an appropriatefusion partner and dimerized complexes thereof) are used to treatpatients who suffer from, for example, autoimmune disorders, fibroticdiseases, inflammatory diseases, ischemic diseases, neurodegenerativediseases, neuropathic diseases, pain disorders, auditory disorders,psychiatric disorders, cancer and trauma and injury.

Examples of autoimmune disorders include, but are not limited to: acutedisseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagicleukoencephalitis, Addison's disease, agammaglobulinemia, alopeciaareata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBMnephritis, antiphospholipid syndrome (APS), autoimmune angioedema,autoimmune aplastic anemia, autoimmune dysautonomia, autoimmunehepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency,autoimmune inner ear disease (AIED), autoimmune lymphoproliferativesyndrome (ALPS), autoimmune myocarditis, autoimmune oophoritis,autoimmune pancreatitis, autoimmune retinopathy, autoimmunethrombocytopenic purpura (ATP), autoimmune thyroiditis, autoimmuneurticaria, axonal & neuronal neuropathies, Balo disease, Behcet'sdisease, cardiomyopathy, Castleman disease, celiac disease, Chagasdisease, chronic fatigue syndrome, chronic inflammatory demyelinatingpolyneuropathy (CIDP), chronic recurrent multifocal ostomyelitis (CRMO),cicatricial pemphigoid/benign mucosal pemphigoid, Cogans syndrome, coldagglutinin disease, congenital heart block, Coxsackie myocarditis, CRESTdisease, Crohn's disease, demyelinating neuropathies, dermatitisherpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica),discoid lupus, Dressler's syndrome, endometriosis, eosinophilicesophagitis, eosinophilic fasciitis, erythema nodosum, essential mixedcryoglobulinemia, Evans syndrome, experimental allergicencephalomyelitis, fibromyalgia, fibrosing alveolitis, giant cellarteritis (temporal arteritis), giant cell myocarditis,glomerulonephritis, Goodpasture's syndrome, granulomatosis withPolyangiitis (GPA) (formerly called Wegener's Granulomatosis), Grave'sdisease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto'sthyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpesgestationis, hypogammaglobulinemia, idiopathic pulmonary fibrosis,idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-relatedsclerosing disease, immunoregulatory lipoproteins, inclusion bodymyositis, interstitial cystitis, juvenile arthritis, juvenile diabetes(Type 1 diabetes), juvenile myositis, Kawasaki disease, Lambert-Eatonsyndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus,ligneous conjunctivitis, linear IgA disease (LAD), Lupus (systemic lupuserythematosus), Lyme disease, chronic, Meniere's disease, microscopicpolyangiitis, mixed connective tissue disease (MCTD), Mooren's ulcer,Mucha-Habermann disease, multiple sclerosis (MS), myasthenia gravis,myositis, narcolepsy, neuromyelitis optica (Devic's), neutropenia,ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism,PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated withStreptococcus), paroxysmal nocturnal hemoglobinuria (PNH), Parry Rombergsyndrome, Pars planitis (peripheral uveitis), Parsonnage-Turnersyndrome, pemphigus, peripheral neuropathy, perivenousencephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritisnodosa, polymyalgia rheumatica, polymyositis, postmyocardial infarctionsyndrome, postpericardiotomy syndrome, primary biliary cirrhosis,primary sclerosing cholangitis, progesterone dermatitis, psoriasis,psoriatic arthritis, pure red cell aplasia, pyoderma gangrenosum,Raynauds phenomenon, reactive Arthritis, reflex sympathetic dystrophy,Reiter's syndrome, relapsing polychondritis, restless legs syndrome,retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis (RA),rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis,scleroderma, Sjogren's syndrome, sperm & testicular autoimmunity, stiffperson syndrome, subacute bacterial endocarditis, Susac's syndrome,sympathetic ophthalmia, Takayasu's arteritis, Temporal arteritis/Giantcell arteritis, thrombocytopenic purpura, Tolosa-Hunt syndrome,transverse myelitis, type 1 diabetes, type I, II, & III autoimmunepolyglandular syndromes, ulcerative colitis, undifferentiated connectivetissue disease (UCTD), uveitis, vasculitis, vesiculobullous dermatosis,vitiligo, and Wegener's granulomatosis.

Examples of fibrotic diseases which may be treated by the scIL-10 andscIL-10 variant peptides (including fusions of each to an appropriatefusion partner) of the invention include, but are not limited to:adhesive capsulitis, arthrofibrosis, atrial fibrosis, chronic kidneydisease, cirrhosis of the liver, cystic fibrosis (CF), Dupuytren'scontracture, endomyocardial fibrosis, glial scar, idiopathic pulmonaryfibrosis, keloid, macular degeneration, mediastinal fibrosis,myelofibrosis, NAFLD/NASH, nephrogenic systemic fibrosis, Peyronie'sdisease, progressive massive fibrosis (lungs), proliferativevitreoretinopathy, pulmonary fibrosis, retroperitoneal fibrosis, scartissue formation resulting from strokes, scleroderma, systemicsclerosis, tissue adhesion.

Examples of inflammatory diseases include, but are not limited to:allergic enteritis, alpha-1-antitrypsin deficiency, ankylosingspondylitis, asthma, Barrett's esophagus, Behcet's disease, chronicfatigue syndrome (CFS/CFIDS/ME), chronic Lyme disease (borreliosis),cocaine-associated vasculitis, Crohn's disease, deficiency of theInterleukin-1 Receptor Antagonist (DIRA), depression, diabetes, FamilialMediterranean Fever (FMF), fibromyalgia (FM), gastroesophageal refluxdisease (GERD), glomerulonephritis, graft versus host disease,granulomatous angiitis, Hashimoto's thyroiditis, hypertension,hyperthyroidism, hypothyroidism, inflammatory bowel disease (IBD),inflammatory myopathies (polymyositis, inclusion body myositis,dermatomyositis), interstitial cystitis (IC), irritable bowel syndrome(IBS), ischemic colitis, kidney stones, Löfgren's syndrome, Lupuserythematosis, methamphetamine-associated vasculitis, migraine headache,Morgellon's, multiple chemical sensitivity (MCS), multiple sclerosis(MS), neonatal onset multisystem inflammatory disease (NOMID), opticneuritis, osteoarthritis, pemphigus vulgaris, polymyalgia rheumatica,prostatitis, psoriasis, psoriatic arthritis, radiation colitis,Raynaud's syndrome/phenomenon, reactive arthritis (Reiter syndrome),reflex sympathetic dystrophy (RSD), restless leg syndrome, rheumatoidarthritis (RA), sarcoidosis, scleroderma, seasonal affective disorder(SAD), septic shock, sinusitis, Sjögren's syndrome, temporal arteritis,tumor necrosis factor (TNF) receptor-associated periodic syndrome(TRAPS), ulcerative colitis, uveitis, vasculitis, and vertigo.

Examples of ischemic diseases include, but are not limited to: acutecoronary syndrome, angina pectoris, angor animi, copeptin, coronaryartery disease, coronary ischemia, hibernating myocardium, ischemicstroke, management of acute coronary syndrome, meldonium, myocardialinfarction, myocardial infarction complications, myocardial infarctiondiagnosis, myocytolysis, post-anoxic encephalopathy, Prinzmetal'sangina, Sgarbossa's criteria, stroke, TIMI, transient ischemic attack(TIA) and unstable angina.

Examples of neurodegenerative diseases include, but are not limited to:ataxia telangiectasia, autosomal dominant cerebellar ataxia,Baggio-Yoshinari syndrome, Batten disease, estrogen andneurodegenerative diseases, hereditary motor and sensory neuropathy withproximal dominance, Infantile Refsum disease, JUNQ and IPOD, locomotorataxia, Lyme disease, Machado-Joseph disease, mental retardation andmicrocephaly with pontine and cerebellar hypoplasia, multiple systematrophy, neuroacanthocytosis, neuronal ceroid lipofuscinosis,Niemann-Pick disease, pontocerebellar hypoplasia, protein aggregation,pyruvate dehydrogenase deficiency, radiation myelopathy, Refsum disease,retinitis pigmentosa, Sandhoff disease, Shy-Drager syndrome, spinalmuscular atrophy, spinocerebellar ataxia, subacute combined degenerationof spinal cord, subacute sclerosing panencephalitis, Tabes dorsalis,Tay-Sachs disease, toxic encephalopathy, toxic leukoencephalopathy andWobbly Hedgehog Syndrome.

Examples of neuropathic diseases include, but are not limited to: Bell'sPalsy, campylobacter-associated motor axonopathies, Charcot-Marie-Tooth,chronic inflammatory demyelinating polyneuropathy, diabetic amyotrophyavulsion, diabetic neuropathies, Guillain Barre Syndrome and vasculitis.

Examples of pain disorders include, but are not limited to: Amplifiedmusculoskeletal pain syndromes, Anterior cutaneous nerve entrapmentsyndrome, central pain syndrome, chronic functional abdominal pain,chronic pain, chronic prostatitis/chronic pelvic pain syndrome, chronicwound pain, degenerative disc disease, dentomandibular sensorimotordysfunction, failed back syndrome, fibromyalgia, interstitial cystitis,irritable bowel syndrome (MS), myofascial pain syndrome, pelvic pain,post-vasectomy pain syndrome, reflex neurovascular dystrophy,sickle-cell disease, theramine, and vulvodynia.

Examples of auditory disorders include, but are not limited to:conductive hearing loss, sensorineural hearing loss (SNHL), mixedhearing loss.

Examples of psychiatric disorders include, but are not limited to: majordepressive disorder, treatment-refractory depression,treatment-resistant depression.

Examples of trauma and injury include, but are not limited to: includingcentral nervous system (CNS) injuries, traumatic brain injury, spinalcord injury, crush injuries, shock, tendon damage, wounds to the cornea,wounds to the eye, skin wounds.

Preferably, an scIL-10 proteins (including fusions of scIL-10 to anappropriate fusion partner and dimerized complexes thereof) of theinvention may be used to treat patients who suffer from, for example,autoimmune disorders including autoimmune lymphoproliferative syndrome(ALPS), autoimmune thyroiditis, Crohn's disease, Grave's disease,Hashimoto's thyroiditis, Kawasaki disease, Lupus (systemic lupuserythematosus), multiple sclerosis (MS), myasthenia gravis, psoriasis,rheumatoid arthritis, Sjogren's syndrome, type 1 diabetes, ulcerativecolitis; fibrotic diseases including Chronic Kidney Disease, cirrhosisof the liver, macular degeneration, NAFLD/NASH, proliferativevitreoretinopathy, pulmonary fibrosis, scar tissue formation resultingfrom strokes, tissue adhesion; including inflammatory diseases includingallergic enteritis, alpha-1-antitrypsin deficiency, asthma, Behcet'sdisease, cocaine-associated vasculitis, glomerulonephritis, Graft VersusHost Disease, granulomatous angiitis, inflammatory bowel disease,inflammatory myopathies (polymyositis, inclusion body myositis,dermatomyositis), ischemic colitis, methamphetamine-associatedvasculitis, optic neuritis, pemphigus vulgaris, radiation colitis,sarcoidosis, Septic Shock, temporal arteritis, vasculitis; ischemicdiseases including myocardial infarction, post-anoxic encephalopathy,stroke; neurodegenerative diseases including neuronal ceroidlipofuscinosis, radiation myelopathy, retinitis pigmentosa, spinalmuscular atrophy; neuropathic diseases includingcampylobacter-associated motor axonopathies, Charcot-Marie-Tooth,chronic inflammatory demyelinating polyneuropathy, diabetic amyotrophyavulsion, diabetic neuropathies, Guillain Barre Syndrome; auditorydisorders including Conductive hearing loss, Sensorineural hearing loss(SNHL), Mixed hearing loss; psychiatric disorders including majordepressive disorder, treatment-refractory depression,treatment-resistant depression; trauma and injury including centralnervous system (CNS) injuries, traumatic brain injury, spinal cordinjury, crush injuries, shock, tendon damage, wounds to the cornea,wounds to the eye, skin wounds.

Most preferably, scIL-10 proteins (including fusions of scIL-10 to anappropriate fusion partner and dimerized complexes thereof) inaccordance with the invention may be used to treat patients who sufferfrom, for example, autoimmune disorders including autoimmunelymphoproliferative syndrome (ALPS), autoimmune thyroiditis, Crohn'sdisease, Grave's disease, Hashimoto's thyroiditis, Kawasaki disease,Lupus (systemic lupus erythematosus), multiple sclerosis (MS),myasthenia gravis, psoriasis, rheumatoid arthritis, Sjogren's syndrome,type 1 diabetes, ulcerative colitis; fibrotic diseases including ChronicKidney Disease, cirrhosis of the liver, macular degeneration,NAFLD/NASH, proliferative vitreoretinopathy, pulmonary fibrosis, scartissue formation resulting from strokes, tissue adhesion; inflammatorydiseases including allergic enteritis, alpha-1-antitrypsin deficiency,asthma, Behcet's disease, cocaine-associated vasculitis,glomerulonephritis, Graft Versus Host Disease, granulomatous angiitis,inflammatory bowel disease, inflammatory myopathies (polymyositis,inclusion body myositis, dermatomyositis), ischemic colitis,methamphetamine-associated vasculitis, optic neuritis, pemphigusvulgaris, radiation colitis, sarcoidosis, Septic Shock, temporalarteritis, vasculitis; ischemic diseases including myocardialinfarction, post-anoxic encephalopathy, stroke; neurodegenerativediseases including neuronal ceroid lipofuscinosis, radiation myelopathy,retinitis pigmentosa, spinal muscular atrophy; neuropathic diseasesincluding campylobacter-associated motor axonopathies,Charcot-Marie-Tooth, chronic inflammatory demyelinating polyneuropathy,diabetic amyotrophy avulsion, diabetic neuropathies, Guillain BarreSyndrome; auditory disorders including Conductive hearing loss,Sensorineural hearing loss (SNHL), Mixed hearing loss; psychiatricdisorders including major depressive disorder, treatment-refractorydepression, treatment-resistant depression; trauma and injury includingcentral nervous system (CNS) injuries, traumatic brain injury, spinalcord injury, crush injuries, shock, tendon damage, wounds to the cornea,wounds to the eye, skin wounds.

Preferably sell-10 proteins (including fusions of scIL-10 to anappropriate fusion partner and dimerized complexes thereof) of theinvention may be used to treat patients who suffer from, for examplecancer of the uterus, cervix, breast, ovaries, prostate, testes, penis,gastrointestinal tract, esophagus, oropharynx, stomach, small or largeintestines, colon, or rectum, kidney, renal cell, bladder, bone, bonemarrow, skin, head or neck, skin, liver, gall bladder, heart, lung,pancreas, salivary gland, adrenal gland, thyroid, brain, gliomas,ganglia, central nervous system (CNS) and peripheral nervous system(PNS), and immune system, spleen or thymus, papilloma virus-inducedcancers, epithelial cell cancers, endothelial cell cancers, squamouscell carcinomas, adenocarcinomas, carcinomas, melanomas, sarcomas,teratocarcinomas, immunogenic tumors, non-immunogenic tumors, dormanttumors, lymphomas, leukemias, myelomas, chemically-induced cancers,metastasis, and angiogenesis, and Tuberous sclerosis.

Preferably, scIL-10 fusion proteins (including fusions of scIL-10 to anappropriate fusion partner and dimerized complexes thereof) inaccordance with the invention may be used to treat patients who sufferfrom auditory disorders, renal cell carcinoma, melanoma, psoriasis,fibrosis, depression, and inflammatory bowel disease (IBD).

Preferably, scIL-10 fusion proteins (including fusions of scIL-10 to anappropriate fusion partner and dimerized complexes thereof) inaccordance with the invention may also be used in the manufacture of amedicament to treat patients to diseases as set forth above, auditorydisorders, auditory disorders, renal cell carcinoma, melanoma,psoriasis, fibrosis, depression, and inflammatory bowel disease (IBD).

The application further provides pharmaceutically acceptablecompositions comprising scIL-10 proteins (including fusions of scIL-10to an appropriate fusion partner and dimerized complexes thereof)described herein. Therapeutic formulations comprising scIL-10 proteinsare prepared for storage by mixing the described proteins having thedesired degree of purity with optional physiologically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980)), in the form of aqueous solutions,lyophilized or other dried formulations. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrans; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulations herein may also contain more than one active compoundsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

The preferably, scIL-10 proteins (including fusions of scIL-10 to anappropriate fusion partner and dimerized complexes thereof) inaccordance with the invention may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the fibronectin based scaffold proteinsdescribed herein, which matrices are in the form of shaped articles,e.g., films, or microcapsules. Examples of sustained-release matricesinclude polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)),polylactides, copolymers of lactide and glycolide, copolymers ofL-glutamic acid and y ethyl-L-glutamate, non-degradable ethylene-vinylacetate, degradable lactic acid-glycolic acid copolymers. While polymerssuch as ethylene-vinyl acetate and lactic acid-glycolic acid enablesustained release of, certain hydrogels release proteins for shortertime periods. When encapsulated proteins remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

While the skilled artisan will understand that the dosage of eachscIL-10 protein (including fusions of scIL-10 to an appropriate fusionpartner and dimerized complexes thereof) in accordance with theinvention will be dependent on the patient's particular circumstances.The dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01to 5 mg/kg, of the host body weight. For example, dosages can be 0.3mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kgbody weight or 10 mg/kg body weight or within the range of 1-30 mg/kg.An exemplary treatment regime entails administration once per week, onceevery two weeks, once every three weeks, once every four weeks, once amonth, once every 3 months or once every three to 6 months. Dosageregimens include 1 mg/kg body weight or 3 mg/kg body weight byintravenous administration, with the protein being given using one ofthe following dosing schedules: every four weeks for six dosages, thenevery three months; every three weeks; 3 mg/kg body weight once followedby 1 mg/kg body weight every three weeks. Preferably, scIL-10 fusionproteins (including fusions of each to an appropriate fusion partner anddimerized complexes thereof in accordance with the invention is usuallyadministered on multiple occasions. Intervals between single dosages canbe, for example, weekly, monthly, every three months or yearly.Intervals can also be irregular as indicated by measuring blood levelsof the soluble protein in the patient. In some methods, dosage isadjusted to achieve a plasma concentration of soluble protein of about0.1-1000 pg/ml and in some methods about 5-300 mg/ml.

For therapeutic applications, scIL-10 proteins (including fusions ofscIL-10 to an appropriate fusion partner and dimerized complexesthereof) in accordance with the invention are administered to a subject,in a pharmaceutically acceptable dosage form. They can be administeredintravenously as a bolus or by continuous infusion over a period oftime, by intramuscular, subcutaneous, intra-ocular, intra-articular,intrasynovial, intrathecal, oral, topical, or inhalation routes. Theprotein may also be administered by intratumoral, peritumoral,intralesional, or perilesional routes, to exert local as well assystemic therapeutic effects. Suitable pharmaceutically acceptablecarriers, diluents, and excipients are well known and can be determinedby those of skill in the art as the clinical situation warrants.Examples of suitable carriers, diluents and/or excipients include: (1)Dulbecco's phosphate buffered saline, pH about 7.4, containing about 1mg/ml to 25 mg/ml human serum albumin, (2) 0.9% saline (0.9% w/v NaCl),and (3) 5% (w/v) dextrose. The methods of the present invention can bepracticed in vitro, in vivo, or ex vivo.

Administration of scIL-10 proteins (including fusions of scIL-10 to anappropriate fusion partner and dimerized complexes thereof), and one ormore additional therapeutic agents, whether co-administered oradministered sequentially, may occur as described above for therapeuticapplications. Suitable pharmaceutically acceptable carriers, diluents,and excipients for co-administration will be understood by the skilledartisan to depend on the identity of the particular therapeutic agentbeing co-administered.

When present in an aqueous dosage form, rather than being lyophilized,scIL-10 (including fusions of scIL-10 to an appropriate fusion partnerand dimerized complexes thereof) typically will be formulated at aconcentration of about 0.1 mg/ml to 100 mg/ml, although wide variationoutside of these ranges is permitted. For the treatment of disease, theappropriate dosage of scIL-10 (including fusions of scIL-10 to anappropriate fusion partner and dimerized complexes thereof) will dependon the type of disease to be treated, the severity and course of thedisease, whether the scIL-10 proteins (including fusions of scIL-10 toan appropriate fusion partner and dimerized complexes thereof) areadministered for preventive or therapeutic purposes, the course ofprevious therapy, the patient's clinical history and response to thescIL-10 protein (including fusions of scIL-10 to an appropriate fusionpartner and dimerized complexes thereof), and the discretion of theattending physician. The scIL-10 protein is suitably administered to thepatient at one time or over a series of treatments.

EXAMPLES Example 1: Unsubstituted scIL-10

Design of scIL-10:C_(L):C_(H)1:Fc and scIL-10:C_(H1):C_(L):Fc

The scIL-10 single chain fusion body molecule contains a covalentlylinked IL-10 homodimer fusion protein linked to the CL-CH1-Fc (Formula3) domain or the CH1-CL-Fc of the IgG1 heavy chain (FIGS. 1 and 2). Theamino acid sequences of each molecule synthesized is found in Table 1.For expression in mammalian cells, the N-terminal leader sequence of SEQID NO: 48 was added to each of the protein sequences found in Table 1.

TABLE 1 Protein Sequence UnsubstitutedSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSscIL-10 (10 aaEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQlinker):CL:CH1:FcEKGIYKAMSEFDIFINYIEAYMTMKIRNGGSGGGGSGGSPGQGTQSENSCTHFPGNLPNMLRDLRD(SEQ ID NO: 12)AFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGGGSGGGGSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSGGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSYLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 12) UnsubstitutedSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSscIL-10 (10 aaEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQlinker):CH1:CL:FcEKGIYKAMSEFDIFINYIEAYMTMKIRNGGSGGGGSGGSPGQGTQSENSCTHFPGNLPNMLRDLRD(SEQ ID NO: 53)AFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGGGSGGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVGGGGSGGGGSGGGGSGGGGSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGSGGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 53) UnsubstitutedSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSscIL-10 (5 aaEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQlinker):FcEKGIYKAMSEFDIFINYIEAYMTMKIRNGGSGGSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRV(Control)KTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKSEQ ID NO: 13)TLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 13)Expression of scIL-10:C_(L):C_(H)1:Fc and scIL-10:C_(H1):C_(L):Fc

The genes were synthetically synthesized and supplied in pcDNA3.1expression vector (GeneArt), and transiently expressed in HEK293 cellsusing the Expi293 expression system (Life Technologies). Proteins werepurified using Protein A (GE Healthcare) with low pH elution anddialyzed against 2 L 1×PBS 2 times.

The molecules were analyzed by SDS PAGE gel under reducing andnon-reducing conditions (FIG. 3). For reducing and non-reducingconditions, 2.5 ug of protein was loaded onto an Any kD gel (Invitrogen)with a Precision Plus Protein Kaleidoscope standard (Invitrogen) (MWrange 10 kD-250 kD). The molecule was characterized by analytical gelfiltration on an) (Bridge Protein BEH SEC column, 200 Å, 3.5 μm, 7.8mm×150 mm (Waters). The column was equilibrated and run at 0.9 ml/minwith 100 mM sodium phosphate pH 7.0 as a running buffer for allanalyses. Purified samples (0.5 mg/ml) were injected (15 ul) and elutedwith a run time of 15 min (FIGS. 4 and 5).

Bioactivity of scIL-10:C_(L):C_(H)1: Fc and scIL-10:C_(H1):C_(L):Fc

In vitro bioactivity was assessed by evaluating the ability ofscIL-10:C_(L):C_(H)1:Fc and scIL-10:C_(H1):C_(L):Fc to stimulateproliferation of the mouse mast cell line MC/9 (ATCC CRL-8306). ThescIL-10 direct Fc fusion protein (scIL-10:Fc) was used as a control. Forthe assay, MC/9 cells were plated at 10,000 cells/well in DMEM mediacontaining 10% heat inactivated fetal bovine serum, 2 mM glutamine and0.05 mM 2-mercaptoethanol. Cells were incubated for 72 hours at 37° C.,5% CO₂ with varying concentrations of human IL-10 (R&D Systems),scIL-10:C_(L):C_(H)1:Fc, scIL-10:C_(H1):C_(L):Fc or scIL-10:Fc. After 72hours, the cells were stained with CellTiter-Blue (Promega) for 4 hoursat 37° C., 5% CO₂ according to the manufacturer's protocol. Fluorescentmeasurements were taken at 560/590 nm. IL-10 (EC₅₀=75 pM),scIL-10:C_(L):C_(H)1:Fc (EC₅₀=79 pM), scIL-10:C_(H1):C_(L):Fc (EC₅₀=93pM) and scIL-10:Fc (EC₅₀=493 pM) were active in a dose dependent fashion(FIG. 6).

Mouse PK of scIL-10:C_(L):C_(H)1: Fc and scIL-10:C_(H1):C_(L):Fc

scIL-10:C_(L):C_(H)1:Fc, scIL-10:C_(H1):C_(L):Fc, and scIL-10:Fcpharmacokinetics in mice were evaluated at a single intravenous doses of0.5 mg/kg administered into tail vein and a single subcutaneous doses of2.5 mg/kg administered into the interscapular region. Blood samples (n=3samples/time point/fusion protein) were collected at 0.083, 0.5, 1, 4,6, 24, 48, 96, 168, 192 and 216 hours after administration ofscIL-10:C_(L):C_(H)1:Fc, scIL-10:C_(H1):C_(L):Fc and scIL-10:Fc. Foreach time point/fusion protein/route of administration, serum was pooledand concentrations were measured using standard MSD techniques.Bioanalytical data was subjected to non-compartmental pharmacokineticanalysis using Phoenix WinNonlin 6.4 software. The pharmacokineticparameter included standard pharmacokinetic parameters of maximumconcentration (C_(max)), time to maximum concentration (T_(max)), areaunder the time versus concentration curve (AUC), mean residence time(MRT), elimination half-life (t1/2), clearance (CL), distribution volumeat steady state (V_(ss)), and bioavailability (% F) were determined andreported in Tables 2 and 3.

TABLE 2 Row Dose Dose Cmax Tmax Cmax/D AUClast ID Compound (mg/kg)(~nMole/kg) ROA (nM) (h) (nM/D) (h*nM) 1 scIL-10:Fc 0.5 3.93 IV 94.90.083 24.2 2080 SEQ ID NO: 13 2 scIL-10:Fc 2.5 19.63 SC 221 24 11.312700 SEQ ID NO: 13 3 scIL-10:C_(L):C_(H)1:Fc 0.5 2.85 IV 140 0.083 49.22850 SEQ ID NO: 12 4 scIL-10:C_(L):C_(H)1:Fc 2.5 14.25 SC 227 24 15.919500 SEQ ID NO: 12 5 scIL-10:C_(H1):C_(L):Fc 0.5 2.84 IV 115 0.083 40.51300 SEQ ID NO: 53 6 scIL-10:C_(H1):C_(L):Fc 2.5 14.2 SC 120 24 8.487570 SEQ ID NO: 53

TABLE 3 CL Row AUCinf AUCinf/D MRTinf t½ (mL/hr/ Vss ID (h*nM) (h*nM)(h) (h) kg) (mL/kg) % F 1 2170 552 33 21 1.811 59.57 NA 2 12700 649 4611 NA NA ~100 3 2850 999 30 7.8 1.001 29.56 NA 4 19500 1370 56 8.5 NA NA~100 5 1300 458 16 9.3 2.183 35.44 NA 6 7570 533 41 9.1 NA NA ~100

Example 2: (scIL-10)-L1-HINGE-Fc Fusion Proteins

Design of scIL-10 Variant Fusion Proteins

The scIL-10 of Formula 1 are fused to a single chain Fc linker ofFormula 2 wherein L1 is CL-CH1-Fc as per Formula 3. The amino acidsequences of each full length scIL-10-L1-HINGE-Fc fusion variant proteinsynthesized is found in Table 4. The description column of Table 4indicates the scIL-10 used in the construct with the fusion partner. Forexample wtIL-10:linker:D41F indicates that in accordance with Formula 1,the first monomer subunit is wt IL-10 of SEQ ID NO: 1 and is thereforeunsubstituted linked to a linker which is in turn linked to the secondmonomer subunit wherein the wtIL10 of SEQ ID NO: 1 is substituted atamino acid 41 such that the isoleucine at amino acid 41 is substitutedwith phenylalanine (D41F).

For expression in mammalian cells, the N-terminal leader sequence of SEQID NO: 48 was added to each of the protein sequences found in Table 4.

TABLE 4 Description Amino Acid Sequence/SEQ ID NO scIL-10:SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF CL:CH1:FcSRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQAENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNKLQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSENSCTHFPGNLP NMLRDLRDAF SRVKTFFQMK DQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 12) M22A:SPGQGTQSEN SCTHFPGNLP NALRDLRDAF linker:D41NSRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA (R1 + R2ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNK mutant)LQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSENSCTHFPGNLP NMLRDLRDAF SRVKTFFQMK NQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 44) D41N:SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF linker:M22ASRVKTFFQMK NQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA (R1 + R2ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNK mutant)LQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSENSCTHFPGNLP NALRDLRDAF SRVKTFFQMK DQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 43) wtIL-10:linker:SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF M22A, D41N SRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA (R1 + R2 ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNK mutant)LQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSENSCTHFPGNLP NALRDLRDAF SRVKTFFQMK NQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 42) wtIL-10:SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF linker:D41NSRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA (R1 mutant)ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNKLQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSENSCTHFPGNLP NMLRDLRDAF SRVKTFFQMK NQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 41) M22A:SPGQGTQSEN SCTHFPGNLP NALRDLRDAF linker:D41ASRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA (R1 + R2ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNK mutant)LQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSENSCTHFPGNLP NMLRDLRDAF SRVKTFFQMK AQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 40) D41A:SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF linker:M22ASRVKTFFQMK AQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA (R1 + R2ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNK mutant)LQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSENSCTHFPGNLP NALRDLRDAF SRVKTFFQMK DQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 39) wtIL-10:linker:SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF M22A, D41ASRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA (R1 + R2 ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNK mutant)LQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSENSCTHFPGNLP NALRDLRDAF SRVKTFFQMK AQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 38) wtIL-10:SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF linker:D41ASRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA (R1 mutant)ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNKLQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSENSCTHFPGNLP NMLRDLRDAF SRVKTFFQMK AQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 37) M22A:SPGQGTQSEN SCTHFPGNLP NALRDLRDAF linker:D41FSRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA (R1 + R2ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNK mutant)LQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSENSCTHFPGNLP NMLRDLRDAF SRVKTFFQMK FQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 29) D41F:SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF linker:M22ASRVKTFFQMK FQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA (R1 + R2ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNK mutant)LQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSENSCTHFPGNLP NALRDLRDAF SRVKTFFQMK DQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 28) wtIL-10:SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF linker:SRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA M22A, D41FENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNK (R1 + R2LQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSEN mutant)SCTHFPGNLP NALRDLRDAF SRVKTFFQMK FQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 27) M22A:SPGQGTQSEN SCTHFPGNLP NALRDLRDAF linker:D41FSRVKTFFQMK FQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA (R1 + R2ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNK mutant)LQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSENSCTHFPGNLP NMLRDLRDAF SRVKTFFQMK DQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 26) M22A: SPGQGTQSEN SCTHFPGNLP NALRDLRDAF linker:M22ASRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA (R2 mutant)ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNKLQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSENSCTHFPGNLP NALRDLRDAF SRVKTFFQMK DQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 25) wtIL-10:linker:SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF M22ASRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA (R2 mutant)ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNKLQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSENSCTHFPGNLP NALRDLRDAF SRVKTFFQMK DQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 24) M22A:SPGQGTQSEN SCTHFPGNLP NALRDLRDAF linker:wtIL-10SRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA (R2 mutant)ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNKLQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSENSCTHFPGNLP NMLRDLRDAF SRVKTFFQMK DQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 23) D41F:SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF linker:D41FSRVKTFFQMK FQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA (R1 mutant)ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNKLQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSENSCTHFPGNLP NMLRDLRDAF SRVKTFFQMK FQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 22) wtIL-10:SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF linkerD41F SRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA (R1 mutant)ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNKLQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSENSCTHFPGNLP NMLRDLRDAF SRVKTFFQMK FQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 21) D41F:linker:SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF wtIL-10SRVKTFFQMK FQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA (R1 mutant)ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNKLQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSENSCTHFPGNLP NMLRDLRDAF SRVKTFFQMK DQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 20) wtIL-10:SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF linker:I87ASRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA (vIL10 mutant)ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNKLQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSENSCTHFPGNLP NMLRDLRDAF SRVKTFFQMK DQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDAKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 19) I87A:linker:SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF wtIL-10SRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA (vIL10 mutant)ENQDPDAKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNKLQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSENSCTHFPGNLP NMLRDLRDAF SRVKTFFQMK DQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 18) I87A: SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF linker:I87ASRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA (vIL10 mutant)ENQDPDAKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNKLQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSENSCTHFPGNLP NMLRDLRDAF SRVKTFFQMK DQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDAKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 17) M22A,SPGQGTQSEN SCTHFPGNLP NALRDLRDAF D41F:linker:SRVKTFFQMK FQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA M22A, D41FENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNK R1 + R2panLQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSEN mutant)SCTHFPGNLP NALRDLRDAF SRVKTFFQMK FQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 30) M22A, D41F:SPGQGTQSEN SCTHFPGNLP NALRDLRDAF linker:M22ASRVKTFFQMK FQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA (R1 + R2ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNK triple mutant)LQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSENSCTHFPGNLP NALRDLRDAF SRVKTFFQMK DQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO:31) M22A, D41F:SPGQGTQSEN SCTHFPGNLP NALRDLRDAF linker:D41FSRVKTFFQMK FQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA (R1 + R2ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNK triple mutant)LQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSENSCTHFPGNLP NMLRDLRDAF SRVKTFFQMK FQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 32) M22A:linker:SPGQGTQSEN SCTHFPGNLP NALRDLRDAF M22A, D41FSRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA (R1 + R2ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNK triple mutant)LQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSENSCTHFPGNLP NALRDLRDAF SRVKTFFQMK FQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO:33) D41F:linker:SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF M22A, D41FSRVKTFFQMK FQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA (R1 + R2ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNK triple mutant)LQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSENSCTHFPGNLP NALRDLRDAF SRVKTFFQMK FQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRNGGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLYSLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 34)

Expression of Sc-IL-10 Variant Fusion Proteins

The genes were synthetically synthesized and supplied in pcDNA3.1expression vector (GeneArt), and transiently expressed in HEK293 cellsusing the Expi293 expression system (Life Technologies). Proteins werepurified using Protein A (GE Healthcare) with low pH elution anddialyzed against 2 L 1×PBS 2 times.

The molecules were analyzed by SDS PAGE gel under reducing andnon-reducing conditions. For reducing and non-reducing conditions, 2.5ug of protein was loaded onto an Any kD gel (Invitrogen) with aPrecision Plus Protein Kaleidoscope standard (Invitrogen) (MW range 10kD-250 kD). The molecule was characterized by analytical gel filtrationon an XBridge Protein BEH SEC column, 200 Å, 3.5 μm, 7.8 mm×150 mm(Waters). The column was equilibrated and run at 0.9 ml/min with 100 mMsodium phosphate pH 7.0 as a running buffer for all analyses. Purifiedsamples (0.5 mg/ml) were injected (15 ul) and eluted with a run time of15 min.

Mouse PBMC Cytokine Release Assay

In vitro bioactivity was assessed by evaluating the ability of ourscIL-10 constructs to inhibit the production of TNFα in LPS stimulatedC57BL/6 mouse PBMCs (Bioreclamation). For the assay, PBMCs cells wereplated at 50,000 cells/well in RPMI media containing 10% heatinactivated fetal bovine serum. Cells were incubated for 18 hours at 37°C., 5% CO₂ with 100 ng/mL LPS and varying concentrations of the scIL-10constructs (R&D Systems). After 18 hours, TNFα production was measuredusing V-Plex mouse TNFa MSD (Mesoscale Discovery). See Tables 5 and 6below for IC50 values.

MC/9 Assay

In vitro bioactivity was assessed by evaluating the ability of ourscIL-10 constructs to stimulate proliferation of the mouse mast cellline MC/9 (ATCC CRL-8306). For the assay, MC/9 cells were plated at10,000 cells/well in DMEM media containing 10% heat inactivated fetalbovine serum, 2 mM glutamine and 0.05 mM 2-mercaptoethanol. Cells wereincubated for 72 hours at 37° C., 5% CO₂ with varying concentrations ofhuman IL-10 (R&D Systems), RDB3515, RDB3516 or RDB3509. After 72 hours,the cells were stained with CellTiter-Blue (Promega) for 4 hours at 37°C., 5% CO₂ according to the manufacturer's protocol. Fluorescentmeasurements were taken at 560/590 nm. See Tables 5 and 6 below for EC50values.

TABLE 5 PBMC + LPS MC/9 SEQ ID NO DESCRIPTION (pM) (pM) RATIO wtIL-100.45 5 11.1 12 scIL-10:CL:CH1:Fc 0.06 60 1000 13 scIL-10:Fc 0.08 4095112.5 14 (scIL-10:Fc), hinge truncation mutant 1 0.06 494 8233.3 15(scIL-10:Fc), hinge truncation mutant 2 0.1 864 8640 16 (scIL-10:Fc),hinge truncation mutant 3 1.4 1007 719.3 17 I87A:linker:I87A (vIL10mutant) 0.38 1775 4671.1 18 I87A:linker:wtIL-10 (vIL10 mutant) 0.03 1073566.7 19 wtIL-10:linker:187A (vIL10 mutant) 0.18 346 1922.2 20D41F:linker:wtIL-10 (R1 mutant) 0.12 264 2200 21 wtIL-10:linkerD41F (R1mutant) 0.18 1368 7600 22 D41F:linker:D41F (R1 mutant) ND ND No activity23 M22A:linker:wtIL-10 (R2 mutant) 0.077 47 610.4 24 wtIL-10:linker:M22A(R2 mutant) 0.045 38 844.4 25 M22A:linker:M22A (R2 mutant) 0.73 541741.1 26 M22A:linker:D41F (R1 + R2 mutant) 2.1 987 470 27wtIL-10:linker:M22A, D41F (R1 + R2 mutant) 2.6 6590 2534.6 28D41F:linker:M22A (R1 + R2 mutant) 8.9 ND >>10000 29 M22A:linker:D41F(R1 + R2 mutant) 4.4 ND >>10000 30 M22A, D41F:linker:M22A, D41F (R1 + R2pan ND ND No activity mutant) 31 M22A, D41F:linker:M22A (R1 + R2 triplemutant) 65 ND >>10000 32 M22A, D41F:linker:D41F (R1 + R2 triple mutant)ND ND No activity 33 M22A:linker:M22A, D41F (R1 + R2 triple mutant) 502ND >>10000 34 D41F:linker:M22A, D41F (R1 + R2 triple mutant) ND ND Noactivity 35 scIL-10:CL:CH1:Fc (scIL10 5aa linker) 0.007 0.6 85.7 36scIL-10:CL:CH1:Fc (scIL10 3aa linker) 0.03 1.5 50

TABLE 6 SEQ ID NO DESCRIPTION PBMC + LPS MC/9 Ratio 12 scIL-10:CL:CH1:Fc0.5 133 266 37 wtIL-10:linker:D41A (R1 mutant) 0.6 1430 2383.333 38wtIL-10:linker:M22A, D41A (R1 + R2 2.4 3602 3602 mutant) 39D41A:linker:M22A (R1 + R2 mutant) 11.1 ND ND 40 M22A:linker:D41A (R1 +R2 mutant) 6 ND ND 41 wtIL-10:linker:D41N (R1 mutant) 1.4 747 533.571442 wtIL-10:linker:M22A, D41N (R1 + R2 0.8 791 988.75 mutant) 43D41N:linker:M22A (R1 + R2 mutant) 4.6 2780 604.3478 44 M22A:linker:D41N(R1 + R2 mutant) 2.1 ND ND

As shown in Table 5, the ratio for WT IL-10 was ˜11. The ratio for SEQID NO: 12 was 1000, showing that just by building the scIL-10 sequenceon the CL:CH1:Fc scaffold, the anti-inflammatory window is increased.The following experiments were conducted with various configurations ofscIL-10 molecules of Formula 1 including unsubstituted scIL-10, scIL-10variants and LINKER lengths of various sizes on the CL:CH1:Fc scaffold.

Experiments were conducted using the constructs of Tables 5 and 6 toexplore the effects unsubstituted scIL-10 and scIL-10 variants thatdisrupt the scIL-10 interfaces with different combinations of the twoIL-10R1 and two IL-10R2 receptor chains from the scIL-10heteropentameric signaling complex. Mutations that disrupt either one ofthe two IL-10R1 interfaces SEQ ID NOS: 20, 21, 37 and 41 as illustratedin FIG. 7, slightly weaken the anti-inflammatory potency, whilesignificantly weakening the immunostimulatory potency, resulting in anincrease in the anti-inflammatory window size.

Introducing a double mutation that simultaneously disrupts both IL-10R1interfaces (SEQ ID NO: 22) results in a construct with no measurableanti-inflammatory or immunostimulatory activities. This demonstratesthat in order for scIL-10 to signal via the IL-10 receptor, it must beable to recruit at least 1 IL-10R1 receptor chain. Since the IL-10R1receptor chain is known to be the “high affinity” receptor chain(binding more tightly to IL-10 than IL-10R2 does), it is likely thatmutations that simultaneously disrupt both IL-10R1 binding interfaceswould eliminate or significantly weaken the ability of scIL-10 to bindto the IL-10 receptor, resulting in no signal transduction at all.

Mutations that disrupt either one of the two IL-10R2 interfaces (SEQ IDNOS: 23 and 24), as illustrated in FIG. 8, demonstrate no change in theanti-inflammatory potency, while showing a slight increase in theimmunostimulatory potency, resulting in slightly decreasedanti-inflammatory window sizes. Introducing a double-mutant thatsimultaneously disrupts both IL-10R2 interfaces (SEQ ID NO: 29) leads toa loss in potency for both anti-inflammatory and immunostimulatoryactivities, resulting in a construct with an anti-inflammatory windowsize similar to that of the IL-10R2 interface single mutants, which isslightly reduced relative to the native scIL-10 construct. This resultdemonstrates that mutations that disrupt the IL-10R2 interface do notalone have the potential to expand the anti-inflammatory window ofscIL-10.

Mutations that simultaneously disrupt one of the IL-10R1 and one of theIL-10R2 interfaces were explored as illustrated in FIG. 8. Mutationslocated in the IL-10R1 and IL-10R2 sites from the same side of thescIL-10 fused dimer (SEQ ID NOS: 26 AND 27) demonstrate weakened potencyfor both anti-inflammatory and immunostimulatory activities; one ofthose combinations (SEQ ID NO: 27) displays a significantly increasedanti-inflammatory window size. Mutations located in an IL-10R1 interfaceand an IL-10R2 interface from opposite sides of the scIL-10 fused dimer(SEQ ID NOS: 28 and 29) display weakened anti-inflammatory potency, andno measurable immunostimulatory activities at the concentrations tested.Therefore, they display extremely large anti-inflammatory windows. SinceIL-10 receptor signal transduction requires IL-10R1 and IL-10R2 to beclustered following IL-10 binding, these data indicate that the optimalstrategy for attenuating immunostimulatory activity (and therebyincreasing the anti-inflammatory window) is to target both of the pairsof IL-10R1/IL-10R2 receptor chains. Since the IL-10R1 interface scanrevealed that signaling requires that at least one IL-10R1 interface becompetent for binding, it is necessary to target the IL-10R2 interfaceon the opposite side of the scIL-10 fused dimer, to effectively disruptboth pairs of IL-10R1/IL-10R2 receptor chains that cluster upon scIL-10binding. This pattern of mutations more dramatically modulates scIL-10bioactivity on cells that mediate immunostimulation, while the cellsthat mediate the anti-inflammatory effects remain quite sensitive toscIL-10 signaling.

Example 3 Varying Linker Length of scIL-10

The scIL-10 of Formula 1 wherein LINKER length is varied are fused to asingle chain Fc linker of Formula 2 wherein L1 is CL-CH1-Fc as perFormula 3. The amino acid sequences of each full lengthscIL-10-L1-HINGE-Fc fusion variant protein synthesized is found in Table7.

For expression in mammalian cells, the N-terminal leader sequence of SEQID NO: 48 was added to each of the protein sequences found in Table 7.

The amino acid sequences of each fusion protein are found in Table 7.Expression of peptides are as described in Example 2. Bioactivity of wastested in a mouse PBMC cytokine release assay and an MC/9 assay asdescribed in Example 2. The results are found in Table 5 of Example 2.The results show that decreasing the size of the linker reduces the sizeof the anti-inflammatory window, implying that the linker length affectsthe strength of the IL-10R1 and IL-10R2 interfaces in ways that reducethe selectivity for anti-inflammatory potency over immunostimulatorypotency.

TABLE 7 Description Amino Acid Sequence UnsubstitutedSPGQGTQSEN SCTHFPGNLP NMLRDLRDAF scIL-10 (5 aaSRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA linker):ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNK CL:CH1:Fc)LQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGSPGQG TQSENSCTHFPGNLPNMLRD LRDAFSRVKT FFQMKDQLDN LLLKESLLED FKGYLGCQALSEMIQFYLEE VMPQAENQDP DIKAHVNSLG ENLKTLRLRL RRCHRFLPCENKSKAVEQVK NAFNKLQEKG IYKAMSEFDI FINYIEAYMT MKIRNGGGGSGGGGSRTVAA PSVFIFPPSD EQLKSGTASV VCLLNNFYPR EAKVQWKVDNALQSGNSQES VTEQDSKDST YSLSSTLTLS KADYEKHKVY ACEVTHQGLSSPVTKSFNRG ECGGGGSGGG GSGGGGSGGG GSASTKGPSV FPLAPSSKSTSGGTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSVVTVPSSSLGT QTYICNVNHK PSNTKVDKRV EPKSCDKTHT CPPCPAPELLGGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVHNAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKTISKAKGQPRE PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNGQPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNHYTQKSLSLSP GK (SEQ ID NO: 39) scIL-10 ofSPGQGTQSEN SCTHFPGNLP NMLRDLRDAF Formula 1SRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA wherein LINKERENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNK is 3 aminoLQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGGSPGQGTQ SENSCTHFPG acid linkerNLPNMLRDLR DAFSRVKTFF QMKDQLDNLL LKESLLEDFK GYLGCQALSEMIQFYLEEVM PQAENQDPDI KAHVNSLGEN LKTLRLRLRR CHRFLPCENKSKAVEQVKNA FNKLQEKGIY KAMSEFDIFI NYIEAYMTMK IRNGGGGSGGGGSRTVAAPS VFIFPPSDEQ LKSGTASVVC LLNNFYPREA KVQWKVDNALQSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC EVTHQGLSSPVTKSFNRGEC GGGGSGGGGS GGGGSGGGGS ASTKGPSVFP LAPSSKSTSGGTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGGPSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTISKAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYTQKSLSLSPGK (SEQ ID NO: 36)

Example 4: Modulating the Anti-Inflammatory Window of scIL-10 Via StericCrowding

The amino acid sequences of each scIL-10 fusion protein used in thisexperiment are found in Table 8. Expression of peptides is as describedin Example 2. Bioactivity of peptides was tested in a mouse PBMCcytokine release assay and an MC/9 assay as described in Example 2. Theresults are found in Table 5 of Example 2. The results show that as thehinge is shortened, the anti-inflammatory window increases in size.Without being limited to any particular theory, this implies that hingetruncation increases steric crowding between two scIL-10 moieties,resulting in modulation of the IL-10R1 and IL-10R2 interfaces, whichtranslates to altered anti-inflammatory and immunostimulatory potencies.

TABLE 8 Description: Amino Acid Sequence scIL-10:FcSPGQGTQSEN SCTHFPGNLP NMLRDLRDAF (5 aa linker)SRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQAENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNKLQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGSPGQG TQSENSCTHFPGNLPNMLRD LRDAFSRVKT FFQMKDQLDN LLLKESLLED FKGYLGCQALSEMIQFYLEE VMPQAENQDP DIKAHVNSLG ENLKTLRLRL RRCHRFLPCENKSKAVEQVK NAFNKLQEKG IYKAMSEFDI FINYIEAYMT MKIRNEPKSSDKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHEDPEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE ALHNHYTQKS LSLSPGK (SEQ ID NO: 13) (scIL-10:FC),SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF 4 AA hingeSRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA truncationENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNKLQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGSPGQG TQSENSCTHFPGNLPNMLRD LRDAFSRVKT FFQMKDQLDN LLLKESLLED FKGYLGCQALSEMIQFYLEE VMPQAENQDP DIKAHVNSLG ENLKTLRLRL RRCHRFLPCENKSKAVEQVK NAFNKLQEKG IYKAMSEFDI FINYIEAYMT MKIRNSDKTHTCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVKFNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVSNKALPAPIEK TISKAKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYPSDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFSCSVMHEALHN HYTQKSLSLS PGK (SEQ ID NO: 14) (scIL-10:Fc),SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF 7 aa hingeSRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA truncationENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNKLQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGSPGQG TQSENSCTHFPGNLPNMLRD LRDAFSRVKT FFQMKDQLDN LLLKESLLED FKGYLGCQALSEMIQFYLEE VMPQAENQDP DIKAHVNSLG ENLKTLRLRL RRCHRFLPCENKSKAVEQVK NAFNKLQEKG IYKAMSEFDI FINYIEAYMT MKIRNTHTCPPCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNWYVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDIAVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ ID NO: 15) (scIL-10:Fc),SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF 10 aa hingeSRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA truncation.ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNKLQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGSPGQG TQSENSCTHFPGNLPNMLRD LRDAFSRVKT FFQMKDQLDN LLLKESLLED FKGYLGCQALSEMIQFYLEE VMPQAENQDP DIKAHVNSLG ENLKTLRLRL RRCHRFLPCENKSKAVEQVK NAFNKLQEKG IYKAMSEFDI FINYIEAYMT MKIRNTHTCPPCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNWYVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDIAVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ ID NO: 16)

Example 5—scIL-10

Experiments were conducted with scIL-10 of Formula 1 wherein LINKER wasof varying lengths. The amino acid sequences synthesized for theseexperiments are shown in Table 9. Expression of SEQ ID NOS: 45 and 46 isas described in Example 2. Bioactivity of SEQ ID NOS 45 was tested in anMC/9 assay as described in Example 2. The data showed that the value forSEQ ID NO: 45 in the MC/9 was 5.6 pM.

Bioactivity of SEQ ID NOS 45 and 46 will be further tested in a mousePBMC cytokine release assay and an MC/9 assay as described in Example 2.The results will show that the scIL-10 moiety, absent any fusionpartner, demonstrates highly potent bioactivity, consistent with thetrends observed for scIL-10 Fc fusion proteins.

TABLE 9 Description Amino Acid Sequence scIL-10 withSPGQGTQSEN SCTHFPGNLP NMLRDLRDAF 5 amino acid SRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA linkerENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNKLQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGSPGQG TQSENSCTHFPGNLPNMLRD LRDAFSRVKT FFQMKDQLDN LLLKESLLED FKGYLGCQALSEMIQFYLEE VMPQAENQDP DIKAHVNSLG ENLKTLRLRL RRCHRFLPCENKSKAVEQVK NAFNKLQEKG IYKAMSEFDI FINYIEAYMT MKIRN (SEQ ID NO: 45)scIL-10 with SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF 10 amino acidSRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA linkerENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNKLQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSENSCTHFPGNLP NMLRDLRDAF SRVKTFFQMK DQLDNLLLKE SLLEDFKGYLGCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHRFLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRN (SEQ ID NO: 46)

Example 6

The scIL-10 of Formula 1 was fused to a mucin domain linker comprising atandem repeat of MUC20 which in turn was fused to an Fc domain. Theamino acid sequence of the (scIL-10 (5aa linker))-(mucin linker)-Fc isfound in Table 10. For expression in mammalian cells, the N-terminalleader sequence of SEQ ID NO: 48 was added to the protein found in Table11.

The amino acid sequences of each fusion protein are found in Table 10.Expression of peptides are as described in Example 2. Bioactivity of wastested in a mouse PBMC cytokine release assay and an MC/9 assay asdescribed in Example 2. The results are found in Table 11. The resultsshow that the bioactivities of scIL-10 Fc fusion proteins are consistentregardless of the composition of the linker domain connecting thescIL-10 and Fc domains.

TABLE 10 Description Amino Acid Sequence (scIL-10(5 aaMYRMQLLSCI ALSLALVTNS SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF linker))-(mucinSRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA linker)-FcENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNKLQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGSGGSPGQG TQSENSCTHFPGNLPNMLRD LRDAFSRVKT FFQMKDQLDN LLLKESLLED FKGYLGCQALSEMIQFYLEE VMPQAENQDP DIKAHVNSLG ENLKTLRLRL RRCHRFLPCENKSKAVEQVK NAFNKLQEKG IYKAMSEFDI FINYIEAYMT MKIRNSGSGGASSESSASSD GPHPVITESR ASSESSASSD GPHPVITESR EPKSSDKTHTCPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKFNWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSNKALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL TCLVKGFYPSDIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSCSVMHEALHNH YTQKSLSLSP GK (SEQ ID NO: 52)

TABLE 11 SEQ ID NO PMBC MC/9 Ratio 52 0.11 pM 21 pM 190.9

Example 7 In Vitro Evaluation of IL-10 Receptor Signaling in Human PBMCs

In vitro bioactivity in a human cell system was assessed by evaluatingthe ability of scIL-10:C_(L):C_(H1):Fc constructs to activate STAT3, asmeasured by intracellular levels of phosphorylated STAT3 protein. Theunmutated scIL-10:C_(L):C_(H1):Fc sequence (SEQ ID NO: 12) was used as acontrol, and constructs containing the receptor-engagement mutationswere used to explore the anti-inflammatory window in human cells. Boththe single R1 mutant (wtIL-10:linker:D41A (SEQ ID NO: 37)) and doubleR1+R2 mutant (wtIL-10:linker:M22A, D41A (SEQ ID NO: 38)) constructs wereused. PBMCs were obtained from BioReclamationIVT, and plated at 4×10⁶cells/well in serum-free media containing (X-vivo 10). Cells wereincubated for 1 hour at 37° C., 5% CO₂, with varying concentrations ofSEQ ID NO: 12, SEQ ID NO: 37, and SEQ ID NO: 38. After 1 hour, cellswere fixed, stained with antibodies for cell surface markers,permeabilized, and stained with antibodies for pSTAT3.

Cell populations were analyzed by flow cytometry. Populations of Bcells, monocytes, NK cells, CD4 T cells, and CD8 T cells were identifiedby cell surface markers (Table 12). Within each population, thepercentage of cells that were positive for pSTAT3 were counted. For eachpopulation, the percent of pSTAT3+ cells was plotted against theconcentration of test article, and a sigmoidal curve fit identified anEC₅₀ for STAT3 activation. The ratio of the EC₅₀ value for monocytes tothe EC₅₀ values for CD4 and CD8 T cells was used to estimate theanti-inflammatory window for each construct.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

While this invention has been particularly shown and described withreferences to preferred features thereof, it will be understood by thoseskilled in the art that various changes in form and details may be madetherein without departing from the scope of the invention encompassed bythe appended claims. It should also be understood that the variousfeatures of the invention described herein are not mutually exclusiveand that features may be combined in whole or in part in accordance withthe invention.

1. A method of modulating the immunostimulatory and anti-inflammatoryproperties of IL-10 in a patient in need of IL-10 therapy comprisingadministering scIL-10 comprising an amino acid sequence arrangement fromN-terminus to C-terminus in accordance with Formula 1:(first monomer subunit)-LINKER-(second monomer subunit)   Formula 1wherein the first monomer subunit, the second monomer subunit or boththe first and second monomer subunits may be independently selectedfrom: an unsubstituted IL-10 monomer subunit; or a substituted IL-10monomer subunit comprising at least one amino acid substitution; whereinscIL-10 modulates the immunostimulatory or anti-inflammatory propertiesas compared to wtIL-10, wherein LINKER is an amino acid linker ofbetween about 1 and about 100 amino acids in length; and wherein scIL-10is optionally covalently attached to a fusion partner.
 2. The method ofclaim 1, wherein LINKER is 5-15 amino acids in length.
 3. The method ofclaim 1, wherein the amino acid substitutions comprise the substitutionof amino acids of scIL-10 that interface with IL-10R1, IL-10R2 or aminoacids that interface with both IL-10R1 and IL-10R2.
 4. The method ofclaim 1, wherein the amino acid substitutions comprise substitutions ofamino acids selected from: methionine at position 22 and aspartic acidat position 41 or any combination thereof.
 5. The method of claim 4,wherein aspartic acid at position 41 is substituted on the first monomersubunit or on the second monomer subunit but not both monomer subunits.6. The method of claim 5, wherein methionine at position 22 issubstituted on only one monomer subunit that is not the same monomersubunit comprising the substitution of aspartic acid at position
 41. 7.The method of claim 1, wherein the amino acid substitution comprisesisoleucine at position
 87. 8. The method of claim 3, wherein the aminoacid substitutions are selected from: methionine at position 22 toalanine (M22A); aspartic acid at position 41 to asparagine (D41N);aspartic acid at position 41 to alanine (D41A); aspartic acid atposition 41 to phenylalanine (D41F).
 9. The method of claim 1,comprising a fusion partner wherein scIL-10 is fused to the hinge regionIgG1.
 10. The method of claim 1, comprising a fusion partner whereinscIL-10 is fused to a modified hinge region if IgG1 wherein themodification to the hinge region is the deletion of between 1 and 10amino acids from the hinge region of IgG1.
 11. The method of claim 1,comprising a fusion partner wherein scIL-10 is fused to the hinge regionof IgG1 via a mucin linker.
 12. The method of claim 11, wherein themucin linker comprises an amino acid sequence that is a tandem repeat ofMUC20.
 13. The method of claim 1, comprising a fusion partner whereinscIL-10 is fused to a single chain Fc linker wherein the fusion proteinhas the sequence of Formula 2(scIL-10)-L1-HINGE-Fc   Formula 2 wherein, L1 is a linker having thefollowing arrangement from amino-terminus to carboxy-terminus:L2-CL-L3-CH1-L4 or L2-CH1-L3-CL-L4 wherein, L2 and L4 are independentlypolypeptide linkers or are independently absent; L3 is a polypeptidelinker; CL is a constant region polypeptide of an immunoglobulin lightchain; and CH1 is a constant region polypeptide from a CH1 domain of animmunoglobulin heavy chain; HINGE is a hinge sequence of animmunoglobulin or is absent with the proviso that if HINGE is absent, L4is present; and Fc is the carboxy-terminus of an immunoglobulin or anyactive fragment or derivative thereof.
 14. An scIL-10 polypeptidecomprising an amino acid sequence arrangement from N-terminus toC-terminus in accordance with Formula 1:(first monomer subunit)-LINKER-(second monomer subunit)   Formula 1wherein the first monomer subunit or the second monomer subunit may beindependently selected from: an unsubstituted IL-10 monomer subunit; ora substituted IL-10 monomer subunit with the proviso that at least oneof the first monomer subunit or the second monomer subunit comprises atleast one amino acid substitution; wherein LINKER is an amino acidlinker of between about 1 and about 100 amino acids in length; andwherein scIL-10 is optionally covalently attached to a fusion partner.15. The polypeptide of claim 14, wherein LINKER is 5-15 amino acids inlength.
 16. The polypeptide of claim 14, wherein the amino acidsubstitutions comprise the substitution of amino acids of scIL-10 thatinterface with IL-10R1, IL-10R2 or amino acids that interface with bothIL-10R1 and IL-10R2.
 17. The polypeptide of claim 14, wherein the aminoacid substitutions comprise substitutions of amino acids selected from:methionine at position 22 and aspartic acid at position 41 or anycombination thereof.
 18. The polypeptide of claim 17, wherein asparticacid at position 41 is substituted on the first monomer subunit or onthe second monomer subunit but not both monomer subunits.
 19. Thepolypeptide of claim 18, wherein methionine at position 22 issubstituted on only one monomer subunit that is not the same monomersubunit comprising the substitution of aspartic acid at position
 41. 20.The polypeptide of claim 14, wherein the amino acid substitutioncomprises isoleucine at position
 87. 21. The polypeptide of claim 16,wherein the amino acid substitutions are selected from: methionine atposition 22 to alanine (M22A); aspartic acid at position 41 toasparagine (D41N); aspartic acid at position 41 to alanine (D41A);aspartic acid at position 41 to phenylalanine (D41F).
 22. Thepolypeptide of claim 1, comprising a fusion partner wherein scIL-10 isfused to the hinge region IgG1.
 23. The polypeptide of claim 14,comprising a fusion partner wherein scIL-10 is fused to a modified hingeregion if IgG1 wherein the modification to the hinge region is thedeletion of between 1 and 10 amino acids from the hinge region of IgG1.24. The polypeptide of claim 14, comprising a fusion partner whereinscIL-10 is fused to the hinge region of IgG1 via a mucin linker.
 25. Thepolypeptide of claim 24, wherein the mucin linker comprises an aminoacid sequence that is a tandem repeat of MUC20.
 26. An scIL-10polypeptide of claim 14, comprising a fusion partner wherein scIL-10 isfused to a single chain Fc linker wherein the fusion protein comprisesan amino acid sequence of Formula 2(scIL-10)-L1-HINGE-Fc   Formula 2 wherein, L1 is a linker having thefollowing arrangement from amino-terminus to carboxy-terminus:L2-CL-L3-CH1-L4 or L2-CH1-L3-CL-L4 wherein, L2 and L4 are independentlypolypeptide linkers or are independently absent; L3 is a polypeptidelinker; CL is a constant region polypeptide of an immunoglobulin lightchain; and CH1 is a constant region polypeptide from a CH1 domain of animmunoglobulin heavy chain; HINGE is a hinge sequence of animmunoglobulin or is absent with the proviso that if HINGE is absent, L4is present; and Fc is the carboxy-terminus of an immunoglobulin or anyactive fragment or derivative thereof.
 27. The polypeptide of claim 26,wherein CL, CH1, HINGE and Fc are at least 90% identical to the CL, CH1,hinge and Fc regions respectively of human IgG1.
 28. The polypeptide ofclaim 26, wherein L3 is a polypeptide linker having the amino acidsequence (GGGGS)_(n) (SEQ ID NO: 54) wherein n is 1-5.
 29. Thepolypeptide of claim 26, wherein L2 is present and is a polypeptidelinker having the amino acid sequence (GGGGS)_(n) (SEQ ID NO: 54)wherein n is 1-5.
 30. The polypeptide of claim 26, wherein L4 is presentand is a polypeptide linker having the amino acid sequence (GGGGS)_(n)(SEQ ID NO: 54) wherein n is 1-5.
 31. The polypeptide of claim 26,wherein HINGE and L2 are present and L4 is absent.
 32. The polypeptideof claim 26, wherein HINGE, L2 and L4 are present.
 33. The polypeptideof claim 26, wherein HINGE is absent and L4 is present.
 34. Thepolypeptide of claim 26, wherein HINGE is absent and L2 and L4 arepresent.
 35. A dimerized complex comprising the polypeptide of claim 26wherein L1 is a linker having the following arrangement fromamino-terminus to carboxy-terminus: L2-CL-L3-CH1-L4.
 36. A polypeptideof claim 26, selected from the group consisting of: SEQ ID NOs: 20-21and SEQ ID NOS: 37-44.
 37. A polypeptide of claim 26, selected from thegroup consisting of: SEQ ID NOS: 17, 18 and
 19. 38. An scIL-10polypeptide comprising an amino acid sequence arrangement fromN-terminus to C-terminus in accordance with Formula 1:(first monomer subunit)-LINKER-(second monomer subunit)   Formula 1wherein the first monomer subunit, the second monomer subunit or boththe first and second monomer subunits may be independently selectedfrom: an unsubstituted IL-10 monomer subunit; or a substituted IL-10monomer subunit; wherein LINKER is an amino acid linker of between about1 and about 100 amino acids in length with the proviso that the linkeris not GGSGGGGSGG (SEQ ID NO: 3); and wherein scIL-10 is covalentlyattached to a fusion partner.
 39. A polypeptide of claim 38, comprisinga fusion partner wherein scIL-10 is fused to a single chain Fc linkerwherein the fusion protein comprises an amino acid sequence of Formula 2(scIL-10)-L1-HINGE-Fc   Formula 2 wherein, L1 is a linker having thefollowing arrangement from amino-terminus to carboxy-terminus:L2-CL-L3-CH1-L4 or L2-CH1-L3-CL-L4 wherein, L2 and L4 are independentlypolypeptide linkers or are independently absent; L3 is a polypeptidelinker; CL is a constant region polypeptide of an immunoglobulin lightchain; and CH1 is a constant region polypeptide from a CH1 domain of animmunoglobulin heavy chain; HINGE is a hinge sequence of animmunoglobulin or is absent with the proviso that if HINGE is absent, L4is present; and Fc is the carboxy-terminus of an immunoglobulin or anyactive fragment or derivative thereof.
 40. A polypeptide of claim 39,selected from SEQ ID NO 35 and SEQ ID NO:
 36. 41. An scIL-10 polypeptidecomprising an amino acid sequence arrangement from N-terminus toC-terminus in accordance with Formula 1:(first monomer subunit)-LINKER-(second monomer subunit)   Formula 1wherein the first monomer subunit, the second monomer subunit or boththe first and second monomer subunits may be independently selectedfrom: an unsubstituted IL-10 monomer subunit; or a substituted IL-10monomer subunit comprising at least one amino acid substitution; whereinLINKER is an amino acid linker of between about 1 and about 100 aminoacids in length; and wherein scIL-10 is covalently attached to a fusionpartner that comprises a mucin domain polypeptide.
 42. The polypeptideof claim 41, comprising SEQ ID NO:
 52. 43. The method of claim 1,wherein the polypeptide comprises SEQ ID NOS: 12-21, 23-29, 31, 33, and35-45 and
 52. 44. The polypeptide of claim 38, wherein the fusionpartner comprises an IgG1 Fc region including a hinge region.
 45. Thepolypeptide of claim 44, comprising SEQ ID NO:
 13. 46. The polypeptideof claim 44, wherein 1-10 amino acids have been deleted from the hingeregion.
 47. The polypeptide of claim 46, selected from SEQ ID NOS:14-16.